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Jul 2021

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Plasma Membrane Wounding and Repair Assays for Eukaryotic Cells
真核细胞的等离子膜损伤和修复试验   

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Abstract

Damage to the plasma membrane and loss of membrane integrity are detrimental to eukaryotic cells. It is, therefore, essential that cells possess an efficient membrane repair system to survive. However, the different cellular and molecular mechanisms behind plasma membrane repair have not been fully elucidated. Here, we present three complementary methods for plasma membrane wounding, and measurement of membrane repair and integrity. The first protocol is based on real time imaging of cell membrane repair kinetics in response to laser-induced injury. The second and third protocols are end point assays that provide a population-based measure of membrane integrity, after either mechanical injury by vortex mixing with glass beads, or by detergent-induced injury by digitonin in sublytic concentrations. The protocols can be applied to most adherent eukaryotic cells in culture, as well as cells in suspension.

Keywords: Cell injury (细胞损伤), Plasma membrane damage (质膜损伤), Membrane wounding (膜损伤), Plasma membrane repair assays (质膜修复检测), Imaging (成像), Membrane integrity (膜完整性)

Background

Unlike prokaryotic cells, which are protected by a cell wall, eukaryotic cells lack this shield, and are thus more vulnerable to membrane lesions (Cooper and McNeil, 2015). The plasma membrane of eukaryotic cells is composed of a phospholipid bilayer with integrated transmembrane proteins, which essentially constitutes the physical barrier separating the cell from the extracellular environment, and sustains an essential osmotic gradient to the outside (Khan et al., 2013). The integrity of the plasma membrane is frequently compromised during the lifetime of most cells by different means. Cells that reside in mechanically active tissue environments, e.g., muscle and lung cells, frequently experience injuries to their plasma membrane (McNeil and Khakee, 1992; Gajic et al., 2003). Both mechanical stresses and chemical stresses, such as pore-forming toxins secreted by invading pathogens (Bischofberger et al., 2009), can each induce membrane damage, which poses an immediate threat to cell survival if not repaired. Hence, cells have developed effective plasma membrane repair mechanisms to cope with membrane injuries and ensure cellular homeostasis. Repair mechanisms are strictly dependent on the influx of extracellular calcium ions (Ca2+) into the cell through the wound, and involve several cellular processes, including cytoskeleton reorganization (Abreu-Blanco et al., 2011b), exocytosis (Bi et al., 1995; Andrews et al., 2014), endocytosis (Idone et al., 2008), and membrane shedding (Scheffer et al., 2014; Jimenez et al., 2014; Sønder et al., 2019). The different repair mechanisms appear to be utilized in combination, depending on the kind of injury imposed on the membrane (Cooper and McNeil, 2015; Boye and Nylandsted, 2016). Furthermore, the influx of extracellular Ca2+ triggers rapid recruitment of various Ca2+-activated repair proteins to the damaged membrane, including members of the annexin family, which are directly involved in the immediate repair response, to seal the hole within seconds (A. K. McNeil et al., 2006; Bouter et al., 2011; Jaiswal et al., 2014; Boye et al., 2017; Sønder et al., 2019). The early repair responses occur within seconds to a few minutes after injury, and both the efficiency and underlying mechanisms of repair determine cellular fate: either cell death, or successful cell repair and restructuring. After initial membrane resealing, where annexins play a major role, cells need to restructure and remove damaged membrane, involving both exocytic and endocytic events, including macropinocytosis. To this end, we recently found that breast cancer cells use macropinocytosis coupled to components of the non-canonical autophagy system (a process termed LC3-associated macropinocytosis), to remove damaged parts of the plasma membrane, and restore membrane integrity (Sønder et al., 2021).


As loss of cell membrane integrity in vivo occurs due to a variety of physiological stressors, several experimental methods have been developed to mimic these conditions. These methods include cell-confined induced injury by passing cells through a narrow bore syringe, scraping attached cells from the substrate, or exposing cell monolayers to rolling glass beads (P. L. McNeil, 2001; P. L. McNeil et al., 2001; Corrotte et al., 2015; Jaiswal et al., 2014). However, these injury methods can be challenging to reproduce exactly, as they depend, for example, on the forces that are applied to the syringe, the cell confluency before scraping, or rolling with glass beads, resulting in great variability in the population of injured cells between samples. On the other hand, more controlled membrane lesions can be obtained using bacterial pore-forming toxins, e.g., Streptolysin O (SLO), which results in approximately 30 nm pores (Tweten, 2005; Idone et al., 2008). However, approaches using pore-forming toxins do not mimic mechanically-induced injuries in vivo, as these toxins chemically alter the cell membrane, by extracting lipids such as cholesterol (Gonzalez et al., 2008; Babiychuk et al., 2011). Thus, the choice of injury type is of great importance, since it can affect what can be learned about the repair response.


The first protocol presented here (Protocol A) is a method to monitor cell membrane repair kinetics in living cells, following UV ablation laser-induced plasma membrane injury. The two following protocols (Protocol B and Protocol C) are end point assays that can be used to identify, e.g., a deficit in the repair ability in different cellular conditions. Laser-induced injury is a very useful experimental approach, as it creates localized and well controlled injuries, which can be combined with live-cell imaging to follow fluorescently tagged proteins during the repair process (Jaiswal et al., 2014; Sønder et al., 2019). The laser injury approach has been used for monitoring cell membrane repair in various studies, in both mammalian and invertebrate organisms (McNeil et al., 2003; Abreu-Blanco et al., 2011a).


To analyze the extent of membrane damage and the kinetics of membrane repair, different fluorescent membrane impermeable dyes can be applied, including Hoechst 33258, propidium iodide (PI), FM1-43, and FM4-64. When the membrane is breached, Hoechst 33258 and PI, which are both membrane impermeant, can enter the cell and bind to nucleic acids. Here, the resulting fluorescence in the nuclei can be quantified as a measure of increased membrane permeability, i.e., poor membrane integrity. The FM dyes are styryl lipophilic dyes that increase in fluorescence intensity upon phospholipid binding. They will enter the cell through a membrane breach, thereby functioning as a read-out of the extent of injury and healing response, from which the repair kinetics can be calculated (Betz et al., 1996; Corrotte et al., 2015). However, FM dyes bind to the plasma membrane and are also taken up by the cell via endocytic mechanisms, which results in intense intracellular staining independent of membrane lesions over the long term. Thus, FM dyes are best suitable for measuring repair kinetics in short term assays. For long term assays, impermeable Hoechst 33258 or PI should be used instead, since these dyes only stain nucleic acids, and do not appear in internalized vesicles, as FM dyes do. For the laser-induced injury protocol, the cell membrane is injured by a high intensity single photon nanosecond pulsed laser, in presence of cell impermeant dye. The injury causes the impermeant dye to enter the cell, and repair restricts further dye entry, resulting in a fluorescence plateau. In contrast, failure to repair causes a continuous entry of the dye into the injured cell, and intracellular dye fluorescence will steadily increase.


The last two protocols presented here are end point assays, and provide a population-based measure for monitoring plasma membrane repair. End point assays are used to gain insight into the involvement of cellular and molecular processes in membrane repair. Their simplicity is an advantage when investigating novel repair proteins, and mechanisms involved in cellular repair, especially when investigating several conditions at the same time. However, the cellular repair response is a complex process that cannot be fully monitored without temporal resolution, which can be achieved using methods that enable controlled local injury of the cell membrane, and allow real time monitoring of the repair response. In both end point assays presented here, membrane integrity is measured using the membrane permeant dye Hoechst 33342, and membrane impermeant dye PI. By using a microscopy-based plate reader (Celigo® Imaging Cytometer), the number of total cells (Hoechst 33342 positive cells) and permeabilized cells (PI positive cells) are measured per well. An advantage of using image-based assays, to quantify membrane permeabilization and cell death, is that it provides information at the single cell level, as compared to enzymatic assays. In the first protocol, cells are mechanically injured by vortex mixing with glass beads (Protocol B), and in the second assay the cells are chemically injured using the detergent digitonin (Protocol C). Digitonin, a saponin from Digitalis purpurea (Sudji et al., 2015), is typically used in most laboratories to completely lyse cell membranes. However, it can also be used in sublytic concentrations, creating plasma membrane damage that can be repaired by cells (Boye et al., 2017; Heitmann et al., 2021). The exact mechanism of digitonin hole formation is still not clear, but known to be dependent on cholesterol in the membrane (Sudji et al., 2015). Therefore, the extent of membrane damage is dependent both on the cholesterol content in the plasma membrane, and digitonin concentration.


With these three protocols, we describe different plasma membrane wounding methods and membrane integrity assays. We have used these methods combined with siRNA knockdown of genes of interest, and pharmacological treatments [e.g., 5-(N-Ethyl-N-isopropyl) amiloride (EIPA), and trifluoperazine (TFP)], to elucidate the role of different repair proteins, including S100A11, and members of the annexin protein family, in membrane repair response to different types of injury (Jaiswal et al., 2014; Sønder et al., 2019; Boye et al., 2017; Heitmann et al., 2021; Sønder et al., 2021).

Protocol A: Plasma membrane repair kinetics upon laser injury in live cells

Materials and Reagents

  1. RPMI 1640, without Phenol Red (Gibco® by Life Technologies, purchased via Thermo Fisher Scientific, catalog number: 32404014)

  2. Trypsin, TrypLETM Express Enzyme (1×), no phenol red (Gibco® by Life Technologies, purchased via Thermo Fisher Scientific, catalog number: 12604013) (storage 4°C)

  3. Fetal Bovine Serum (FBS) (Gibco® by Life Technologies, purchased via Thermo Fisher Scientific, catalog number: 10270106, 6%, storage 4°C)

  4. GlutaMax (Gibco® by Life Technologies, purchased via Thermo Fisher Scientific, catalog number: 35050061, 2 mM, storage 4°C)

  5. HEPES buffer solution stock 1 M (Sigma-Aldrich, catalog number: H3375, dissolved in H2O, storage 4°C)

  6. Calcium-free imaging media (e.g., Hanks’ balanced salt solution (HBSS), without calcium, magnesium, nor phenol red (Gibco® by Life Technologies, purchased via Thermo Fisher Scientific, catalog number: 14175095, storage 4°C, use pre-heated to 37°C)

  7. Cells tested in the current protocol

    1. MCF7 human breast carcinoma cells

    2. HeLa human cervix carcinoma cells

    3. SH-SY5Y is a thrice-subcloned human cell line derived from the SK-N-SH neuroblastoma cell line

  8. FM1-43 (Invitrogen, catalog number: T3163, 1.6 µM, store in aliquots -20°C)

  9. FM4-64 (Invitrogen, catalog number: T13320, 2.5 µM, store in aliquots -20°C)

  10. Propidium iodide (PI) (Sigma-Aldrich, catalog number: P4864, 0.5 µg/mL, stock 1 mg/mL, dissolved in H2O, storage 4°C)

  11. Hoechst 33258 (Sigma-Aldrich, catalog number: 861405, 2.5 µg/mL, storage 4°C)

  12. Cell imaging media (CIM) (see Recipes)

Equipment

  1. Cell culture incubator set at 37°C, 5% CO2

  2. 35 mm, No. 1.0 Coverslip, 14 mm glass bottom, uncoated MatTek dish (MatTek Corporation, p35G-1.0-14-C)

  3. Confocal microscope equipped with a spinning disk, ablation laser and a 63× water objective. In this protocol we use the following microscope and equipment:

    1. Inverted microscope Eclipse Ti-E (Nikon) with a 63× water objective

    2. UltraVIEW VoX Spinning Disk (PerkinElmer)

    3. 355 nm UV ablation laser (Rapp OptoElectronic)

    4. Heated chamber (37°C) for live cell imaging

Software

  1. Volocity software (PerkinElmers)

  2. SysCon software (Rapp OptoElectronic)

  3. Prism (GraphPad Software, Inc) or Microsoft Excel (Microsoft)

Procedure

A. Real time kinetics of plasma membrane repair following laser injury using live cell imaging

  1. Seed 1 × 105 cells in a 35-mm MatTek imaging-culture dish with a glass bottom, and allow cells to adhere overnight in a cell culture incubator at 37°C.

    Set up 2–3 dishes per condition. Depending on the cell line, the number of seeded cells should be adjusted to obtain approximately 50% confluency on the day of the experiment.

  2. Optional step: Pretreat cells with an inhibitor, or apply siRNA to knockdown a gene of interest.

  3. Next day (on the day of experiment) prepare imaging media: preheat cell imaging media (CIM, see Recipes) at 37°C.

  4. Prepare FM dye solution in preheated CIM.

    The optimal dye concentration varies between dyes and cell lines, and should be optimized. For MCF7 and HeLa cells, FM1-43 at 1.6 µM works well. In this example, we use the FM1-43 dye in MCF7 cells.

    Note: Depending on your experimental setup, other impermeable dyes, such as FM4-63, impermeable Hoechst 33258, or PI, can be used as well.

  5. Wash cells with preheated CIM, and add 1–2 mL of CIM containing the FM dye to the dish.

  6. Place the dish in the microscope holder in the stage maintained at 37°C, and set the focus to visualize plasma membrane-associated FM dye (Figure 1A; and Videos 1 & 2). We use a 63× water objective in an inverted microscope Eclipse Ti-E (Nikon) equipped with a top incubator, to maintain a steady temperature.

    Note: The following steps provide a general outline of the experiment. Details will change depending on the microscope used, so the user should get training for their particular setup before performing the experiment.

  7. Select a 1–2 µm region of interest within the plasma membrane of any intact single cell, orient the laser to that region, and irradiate with a 355 nm UV ablation laser for <4 ns, using an optimal laser power. The laser power of the instrument should be adjusted, to obtain a reproducible and non-lethal injury.

    Notes:

    1. The ablation laser is always calibrated before starting the experiments. Settings of the ablation laser are instrument- and cell line-dependent, and must be optimized. In our laboratory, we use the following setting at our system for MCF7 and HeLa cells: 2.6% power, 200 Hz repetition rate, pulse energy >60 µJ, and pulse length <4 ns.

    2. Our system allows making laser stripes, rectangles, and spot injuries. We use the spot laser injury consistently (laser injury site indicated by arrow in Figure 1A, or star on videos), with the laser settings indicated above. However, in other systems, e.g., 3i laser ablation system, the injury area can be adjusted at will.

    3. When working with lasers, you should always have laser safety in place, especially when working with pulsed lasers, as these can reach very high power in each short pulse, making them very dangerous. Some safety precautions include having an incubation chamber with laser interlock, and laser safety goggles, which should be used when there is a risk of potential exposure to the eyes.

  8. Monitor the repair kinetics by imaging every 5 s in bright field and epifluorescence, starting prior to injury, and continuing for 5 min following injury.

    For a negative control (i.e., no repair), repeat steps A4–A8 using Ca2+-free CIM containing the FM dye. Repeat steps A7–A8 for at least 10 cells per condition, obtained in three independent experiments.

    Note: If the assay is combined with any pre-treatment, the researcher should include samples with control cells without laser injury, to rule out plasma membrane injury resulting from the pre-treatment alone.

  9. Images are acquired with the inverted microscope Eclipse Ti-E (Nikon) paired with the UltraVIEW VoX Spinning Disk (PerkinElmer), using the 63× water objective.

Data analysis

Representative data

Protocol A describes how to measure the kinetics of plasma membrane repair upon laser injury and subsequent repair in live cells, by monitoring the entry of FM dye in the cells.

  1. To quantify plasma membrane permeability (a read-out of membrane integrity), measure the intracellular FM dye fluorescence intensity at a single cell level across time points, using appropriate imaging software, e.g., Volocity. The intracellular FM dye fluorescence intensity is measured by selecting a specific region of interest inside the cell at the site of injury (the region where FM dye starts entering the cell: area outlined by the white dashed line in Figure 1A). Calculate the change in fluorescence intensity (F/F0) during the course of imaging, by normalizing the fluorescence intensity for each time point (F) to the fluorescence intensity before injury (F0). At least 10 cells per condition should be measured.



    Figure 1. Example of real time imaging of cell membrane repair in response to laser injury.

    (A) MCF7 cells were injured by ablation with an UV laser in media containing FM1-43, and in the presence (top panel), or absence (bottom panel) of Ca2+. Representative images of injured MCF7 cells showing intracellular FM1-43 accumulation over time (before and after the injury). White arrows indicate injury sites, while yellow arrows indicate FM1-43 accumulation. The area outlined by the white dashed line illustrates the area used for FM1-43 fluorescence intensity quantification. See Videos 1 & 2. (B) Quantification of single cell membrane repair kinetics upon laser injury in a MCF7 cell, in media with or without Ca2+, measured as the change in FM1-43 dye fluorescence intensity (F/F0) over time, in the region where FM1-43 dye enters the cell (site of damage). The black arrow indicates the injury time point. Scale bar =4.6 µm.


  2. Plot the mean or individual cell changes in fluorescence intensity (F/F0: y-axis) over time (x-axis) (Figure 1B). When representing as means, at least 10 cells per condition obtained from three independent experiments must be considered, and the standard error of mean (SEM) must be plotted. For statistical analysis, area under the curve (AUC) analysis followed by unpaired t-test with Welch’s correction are calculated. For example of quantification of more cells, and a plot representing mean fluorescence intensity, see Figure 1A in Sønder et al. (2021).

  3. Representative images for each condition are selected at different time points starting before injury, to assemble a panel of images showing the influx of FM dye under the different conditions (with or without Ca2+) (Figure 1A). The representative images are annotated to demonstrate how the region of interest is selected (Figure 1A).


    Video 1. Uptake of FM1-43 dye after laser injury in media with Ca2+ in a MCF7 cell


    Video 2. Uptake of FM1-43 dye after laser injury in media without Ca2+ in a MCF7 cell

Notes

The FM dyes bind to lipid membranes, but does not diffuse across intact plasma membranes. However, FM dyes are also taken up by the cell via endocytosis, making the assay for measuring repair kinetics only suitable for short term assays. The timescale for performing the experiment depends on how fast the cell of choice is taking up the FM dye without any damage. In our laboratory, where we use MCF7 or HeLa cells, we normally only use a dish for laser-induced injury experiments for 20–30 min after addition of FM-dye; after this time, it becomes difficult to measure the additional influx of FM-dye after injury.

For long term assays, impermeable Hoechst 33258 or PI should be used. The assay can also be combined with different pre-treatments, such as trifluoperazine (TFP), or knockdown of a gene of interest by siRNAs. For examples where the assay is combined with different pre-treatments, see Sønder et al. (2019), Heitmann et al. (2021), and Sønder et al. (2021).

Recipes

  1. Cell imaging media (CIM)

    Colorless RPMI 1640 supplemented with 6% FBS, 2 mM GlutaMax, and 25 mM HEPES.

Protocol B: Membrane integrity following mechanical injury using vortex mixing with glass beads

Materials and Reagents

  1. 6-well cell culture plates or 10-cm cell culture dishes

  2. Eppendorf tubes

  3. Falcon tubes

  4. 96-well cell culture plates clear flat bottom

  5. Multichannel pipette

  6. RPMI 1640 (Gibco® by Life Technologies, purchased via Thermo Fisher Scientific, catalog number: 11875085)

  7. Fetal Bovine Serum (Gibco® by Life Technologies, purchased via Thermo Fisher Scientific, catalog number: 10270106) (6%, storage 4°C)

  8. Hanks’ balanced salt solution (HBSS), no calcium, no magnesium, no phenol red (Gibco® by Life Technologies, purchased via Thermo Fisher Scientific, catalog number: 14175095) (storage 4°C, use pre-heated to 37°C)

  9. Trypsin, TrypLETM Express Enzyme (1×), no phenol red (Gibco® by Life Technologies, purchased via Thermo Fisher Scientific, catalog number: 12604013) (storage 4°C)

  10. DPBS, no calcium, no magnesium (Gibco® by Life Technologies, purchased via Thermo Fisher Scientific, catalog number: 14190144) (storage 4°C)

  11. Cells tested in the current protocol

    1. MCF7 human breast carcinoma cells

    2. HeLa human cervix carcinoma cells

    3. MDA-MB-231 cells, originating from breast carcinoma

  12. 425-600 µm glass beads (Sigma-Aldrich, catalog number: G8772) (Storage RT)

  13. bisBenzimide H 33342 trihydrochloride (hereafter Hoechst, catalog number: 33342) [Sigma-Aldrich, catalog number: B2261, 6.25 µg/mL, stock (25 mg/mL), dissolved in H2O}. Store at -20°C long term, and at 4°C for a short time, where it is stable for 1 month.

  14. Propidium iodide (PI) [Sigma Aldrich, P4864, 0.25 µg/mL, stock (1 mg/mL), dissolved in H2O]. Store at 4°C.

Equipment

  1. Balance

  2. Cell culture incubator set at 37°C, 5 % CO2

  3. V-shape reservoirs

  4. Vortex mixer

  5. Timer

  6. Plate-based bright field and fluorescent imaging cytometer, with minimum two fluorescent channels (excitation 377/50 and emission 477/22, and excitation 531/40 and emission 629/53), e.g., Celigo® Imaging Cytometer (Brooks Life Science Systems).

Software

  1. Celigo Software (Brooks Life Science Systems)

  2. Prism (GraphPad Software, Inc), or Microsoft Excel (Microsoft)

Procedure

B. Membrane integrity following mechanical injury using vortex mixing with glass beads

Note: All cell culture incubations should be performed in a humidified 37°C, 5% CO2 incubator, unless otherwise specified. See Figure 2A for flowchart of the protocol.

  1. Prepare 1.5-mL Eppendorf tubes with 250 mg glass beads (425–600 µm).

    Note: Prepare at least three Eppendorf tubes per cell line per condition.

  2. Seed an appropriate amount of cells in either a 6-well culture plate, or 10-cm culture dish, depending on the experiment.

    Note: The cells should not be more than 80% confluent on the day of the experiment.

  3. Optional step: Pretreat cells with an inhibitor, or perform siRNA knockdown of a gene of interest.

  4. On the day of experiment, trypsinize adherent cells of interest.

    1. Remove all media from the cell culture dish.

    2. Wash the adherent cell monolayer once, with a small volume of DPBS without Ca2+ and Mg2+, to remove any residual FBS that may inhibit the action of trypsin.

      Note: Wash cells in a buffered salt solution that is Ca2+- and Mg2+-free, as Ca2+ and Mg2+ in the salt solution can cause cells to stick together.

    3. Add enough trypsin to the cell culture, to cover the adherent cell layer.

    4. Incubate cells in a cell culture incubator for 2–5 min.

    5. Tap the bottom of the plate on the countertop to dislodge cells.

      Note: Check the cell culture with an inverted microscope to be sure that cells are rounded up and detached from the surface. If cells are not sufficiently detached, return the cell culture to the incubator for an additional minute or two.

    6. Add appropriate culture media, and resuspend the cells.

  5. Count cells and separate them into two falcon tubes, one tube for cells in media with Ca2+, and one tube for cells in media without Ca2+

    Note: If the cells have been pretreated with an inhibitor or by siRNA knockdown of a gene of interest, they should be counted for each condition.

  6. Spin the cells down at 300 × g at room temperature for 4 min, and resuspend cells in media, either with or without Ca2+. HEPES is added to both falcon tubes to a final concentration of 25 mM.

  7. Load 7 × 104 cells in suspension with 250 mg glass beads (425–600 µm) in a 1.5-mL Eppendorf tube, at a density of 200,000 cells/mL.

    Notes:

    1. The number of cells should be optimized for different cell lines. The appropriate number of cells needs to be loaded to the glass beads in a volume of 350–400 µL. For MCF7 and HeLa cells, we use 7 × 104 cells in suspension with 250 mg glass beads (425–600 µm), loading 350 µL resuspended cells at a density of 200,000 cells/mL on the glass beads.

    2. As negative control of no repair, include samples with cells in Ca2+-free media loaded on glass beads.

  8. Incubate tubes in a cell culture incubator for 10 min.

    Note: The cells are incubated to maintain optimal cell conditions, including temperature (37°C), before injury by vortex mixing.

  9. Meanwhile, add 50 µL of media, either with or without Ca2+, to the respective wells in a 96-well plate.

  10. Injure the cells by vortex mixing at maximum speed for 0, 30, or 60 s.

    The vortex time might need optimization, as some types of cells are more fragile than others, and the repair efficiency also varies between different cell lines. The optimal vortex time should result in 10–30% permeabilized cells in media with Ca2+ without any other pre-treatment, since most cells need to be able to repair their membrane damage in this condition. This balance results from the fact that a certain amount of cells need to be permeabilized to ensure that the cells have been damaged, but, on the other hand, there should still be room for an increase in permeabilized cells, when other conditions where the cells are compromised in their membrane repair are included in the assay.

  11. Immediately after injury, plate 10,000 cells per well in a 96-well plate. Make triplicates for each condition. Shake the plate by hand to distribute the cells in the wells.

    Note: Each 96-well should contain 50 µL of media with resuspended cells (10,000 cells), and the 50 µL culture media added in step B9.

  12. Incubate the 96-well plate in a cell culture incubator for 5 min, for the cells to repair.

  13. Prepare PI and Hoechst 33342 dye mix, in media with and without Ca2+.

    Note: Final concentrations should be 0.25 µg/mL PI, and 6.25 µg/mL Hoechst 33342.

  14. Carefully add 50 µL of the respective PI/Hoechst dye mix to the respective wells in the 96-well plate, and incubate in a cell culture incubator for 5 min.

  15. Measure membrane integrity using an imaging cell cytometer (e.g., Celigo®), by determining membrane integrity using the number of total cells (Hoechst 33342 positive cells: excitation 350 nm, emission 461 nm), and the number of permeabilized cells (PI positive cells: excitation 535 nm, emission 617 nm) per well.

    The Celigo® is a multichannel imaging cell cytometer that can be used to perform whole-well live cell analysis, using optical microscopy. In this experiment, cells are labeled with permeable Hoechst 33342 dye, and impermeable PI dye, prior to scanning and analysis. The cytometer scans the selected area, e.g., a 96-well in the selected channels. The software can then be used to identify labeled cells in the selected channel, thereby calculating the number of total cells (Hoechst positive cells), and the number of permeabilized cells (PI positive cells).

    Note: The user should get particular training in their equipment before setting up the experiment.

Data analysis

Representative data

Protocol B describes how to monitor membrane integrity after mechanical-induced injury by vortex mixing with glass beads.

  1. Calculate the mean values of the triplicate measurements for PI (permeabilized cells) and Hoechst 33342 (total cells) positive cells.

  2. Calculate the percentage of permeabilized cells per condition, by relating the total number of PI positive cells to the total number of Hoechst 33342 positive cells per condition. For statistical analysis, at least three independent experiments should be performed, and standard deviations should be plotted. For examples where statistical analysis has been applied, and the assay is combined with different pre-treatments, see Heitmann et al. (2021), and Sønder et al. (2021).



    Figure 2. Example of membrane integrity assay, following mechanically-induced injury by vortex with glass beads.

    (A) Flowchart of the protocol. (B) Membrane integrity assay sample setup in a typical 96-well format in media with and without Ca2+. (C) Plasma membrane integrity in MCF7 cells in suspension exposed to vortex with glass beads for 0, 0.5, or 1 min, in media with or without Ca2+. The percentage of permeabilized cells is calculated based on the number of cells containing impermeable PI, and the total number of cells (detected by the permeable Hoechst 33342 dye). Data represent means of triplicate measurements, and error bars indicate SD values. The optimal vortex time is the time that results in 10–30% permeabilized cells for cells in media with Ca2+ without any other pre-treatment, since most cells need to be able to repair their membrane damage in this condition. Here, 1 min of vortex mixing is the optimal vortex time for MCF7 cells, as this results in approximately 12% permeabilized cells in media with Ca2+ after vortex mixing with glass beads.

Notes

The assay can also be combined with different pre-treatments, e.g., inhibitors of different biological processes, such as 5-(N-Ethyl-N-isopropyl) amiloride (EIPA), that inhibits macropinocytosis, or e.g., knockdown of a gene of interest by siRNAs. For examples where the assay is combined with different pre-treatments, see Heitmann et al. (2021), and Sønder et al. (2021).

Protocol C: Membrane integrity following detergent induced injury using digitonin

It should be noted that the ability of digitonin to permeabilize cellular membranes depends not only on the digitonin concentration, but also on the total amount of digitonin molecules per cell. Thus, digitonin should always be used in the same volume per cell.

Materials and Reagents

  1. Eppendorf tubes

  2. 96-well cell culture plates clear flat bottom

  3. Multichannel pipette

  4. RPMI 1640 (Gibco® by Life Technologies, purchased via Thermo Fisher Scientific, 11875085)

  5. Fetal Bovine Serum (Gibco® by Life Technologies, purchased via Thermo Fisher Scientific, catalog number: 10270106) (6%, storage 4°C)

  6. Cells tested in the current protocol

    1. MCF7 human breast carcinoma cells

    2. HeLa human cervix carcinoma cells

    3. NCI-H1299 originating from non-small-cell lung cancer

    4. MDA-MB-231 cells originating from breast carcinoma

  7. Digitonin (Sigma-Aldrich), Digitonin Stocks (5 and 50 mg/mL), dissolved in H2O. Store at -20°C. Digitonin usually precipitates, and must be redissolved by heating and occasional vortexing. Immediately before use, heat the digitonin stock to 95°C for 5 min, to dissolve precipitates.

  8. Hoechst 33342 (Sigma-Aldrich, catalog number: B2261, 6.25 µg/mL, stock (25 mg/mL), dissolved in H2O. Store at -20°C long term, and 4°C short time (stable for 1 month).

  9. Propidium iodide (PI) (Sigma-Aldrich, catalog number: P4864, 0.25 µg/mL, stock (1 mg/mL), dissolved in H2O. Store at 4°C.

Equipment

  1. Cell culture incubator set at 37°C, 5% CO2

  2. V-shape reservoirs

  3. Vortex mixer

  4. Heat block

  5. Timer

  6. Plate-based bright field and fluorescent imaging cytometer, with a minimum of two fluorescent channels (excitation 377/50 and emission 477/22, and excitation 531/40 and emission 629/53), e.g., Celigo® Imaging Cytometer (Brooks Life Science Systems)

Software

  1. Celigo Software (Brooks Life Science Systems)

  2. Prism (GraphPad Software, Inc) or Microsoft Excel (Microsoft)

Procedure

C. Membrane integrity following detergent induced injury using digitonin

Part 1: Determining the optimal digitonin concentration for creating plasma membrane damage that most cells are able to repair (see Figure 3A for flowchart of the protocol).

The following procedure is necessary to determine the optimal digitonin concentration for creating plasma membrane damage that can be repaired by most cells (~80%) in Ca2+ containing media, without any pre-treatments. The procedure should be performed regularly, as reagents (e.g., digitonin stocks) and cellular conditions may change over time. Performing this procedure ensures consistency of the number of injured cells, and reproducibility of the results obtained. The procedure should be performed for each cell line separately.

  1. Seed 6 × 103 cells per well in 100 µL of culture media in a 96-well plate, and allow cells to adhere overnight in a cell culture incubator at 37°C. Make two plates: one plate is used for determining optimal digitonin concentration, and the other plate is used for measuring membrane integrity.

    Adjust the number of cells per well for each cell line, as the cells need to be 50% confluent on the day of the experiment.

  2. Immediately before use, heat up the digitonin stock (5 µg/µL) to 95°C for 5 min to dissolve any precipitates.

    Note: Digitonin usually precipitates and needs to be redissolved by heating and occasional mixing by vortexing.

  3. Prepare the following ten dilutions of digitonin in preheated culture media (37°C): 0, 5, 7.5, 10, 12.5, 15, 20, 25, 30, and 100 µg/mL digitonin,

    Note: A digitonin solution of 100 µg/mL is used for total/complete permeabilization of cells.

  4. Add 100 µL of digitonin dilutions per well in triplicates. Add the digitonin solution to the side of the well to avoid detachment of adherent cells.

  5. Incubate in a cell culture incubator at 37°C for 30 min. In the meanwhile, prepare media with PI and Hoechst 33342 dye dilutions; the final concentrations should be: 0.25 µg/mL PI, and 6.25 µg/mL Hoechst 33342.

  6. Carefully add 50 µL of PI/Hoechst dye mix to each well in the 96-well plate, and incubate in a cell culture incubator for 5 min.

  7. Measure membrane integrity using the Celigo® cytometer, by measuring the number of total cells [Hoechst 33342 (excitation 350 nm, emission 461 nm) positive cells] and the number of permeabilized cells [PI (excitation 535 nm, emission 617 nm) positive cells] per well.

  8. Determine optimal digitonin concentration.

    1. Calculate the percentage of PI positive cells for the different conditions. The digitonin concentration that enables approximately 80% of the cells to repair [i.e., around 20 % of permeabilized cells (PI positive)] is optimal.

      In this example, where MCF7 cells are treated with digitonin for 30 min, an optimal digitonin concentration is 7.5–10 µg/mL (see Figure 3C).


Part 2: Measuring membrane integrity following detergent induced injury using digitonin

In the following procedure, the membrane integrity is measured for cells treated with digitonin in media with or without Ca2+. The assay can also be combined with different pre-treatments, such as trifluoperazine (TFP), or knockdown of a gene of interest by siRNAs. For examples where the assay is combined with different pre-treatments, see Sønder et al. (2019), and Heitmann et al. (2021). See Figure 4A for a flowchart of the protocol.

  1. Optional step: pretreat cells with an inhibitor.

  2. Prepare digitonin solutions (using the optimal concentration obtained in step C8) in media containing Ca2+ and in Ca2+-free media. Immediately before use, heat up the digitonin stock (5 µg/µL) to 95°C for 5 min, to dissolve any precipitates.

    Notes:

    1. Digitonin usually precipitates and needs to be redissolved by heating and occasional mixing by vortexing.

    2. As negative control (i.e., no repair) include samples with cells in Ca2+-free media.

  3. Wash cells twice in HBSS without Ca2+. In the final wash, only add HBSS without Ca2+ to the wells that will be treated with digitonin in media without Ca2+. The rest of the wells should contain normal culture media.

  4. Add 100 µL of each digitonin dilution per well, in triplicates, as descripted in step C4. Add the digitonin solution to the side of the well to avoid detachment of adherent cells.

  5. Incubate in a cell culture incubator at 37°C for 30 min. In the meanwhile, prepare PI and Hoechst 33342 dye dilutions, in media with and without Ca2+. The final concentrations should be 0.25 µg/mL PI, and 6.25 µg/mL Hoechst 33342.

  6. Carefully add 50 µL of the respective PI/Hoechst dye mixes to the respective wells in the 96-well plate, and incubate in a cell culture incubator for 5 min.

  7. Measure membrane integrity using an imaging cell cytometer (e.g., Celigo®) by determining membrane integrity using the number of total cells (Hoechst 33342 positive cells: excitation 350 nm, emission 461 nm) and the number of permeabilized cells (PI positive cells: excitation 535 nm, emission 617 nm) per well.

    The Celigo® is a multichannel imaging cell cytometer that can be used to perform whole-well live cell analysis using optical microscopy. In this experiment, cells are labeled with permeable Hoechst 33342 dye and impermeable PI dye, prior to scanning and analysis. The cytometer scan the selected area, e.g., a 96-well in the selected channels. Then, the software can be used to identify labeled cells in the selected channel, and thereby calculating the number of total cells (Hoechst positive cells) and the number of permeabilized cells (PI positive cells).

    Note: The user should get particular training in their equipment before setting up the experiment.

Data analysis

Representative data

Protocol C describes how to monitor membrane integrity of cells after chemical-induced injury by the detergent digitonin. First, the optimal digitonin concentration for creating plasma membrane damage that can be repaired by the cells is determined.

  1. Calculate the mean values of the triplicate measurements for PI (permeabilized cells) and Hoechst (total cells) positive cells per condition.

  2. Calculate the percentage of permeabilized cells per condition, by relating the total number of PI positive cells to the total number of Hoechst positive cells. For statistical analysis, at least three independent experiments should be performed, and standard deviations should be plotted. For examples where statistical analysis has been applied, and the assay is combined with a pre-treatment, see Heitmann et al. (2021).

  3. Representative images for each condition are selected to assemble a panel of images showing the incorporation of Hoechst 33342 and PI dye at a single cell level in the presence and absence of Ca2+, before and after digitonin treatment (Figure 4D).



    Figure 3. Example of an optimization experiment, used to determine the optimal digitonin concentration for a membrane integrity assay using MCF7 cells.

    (A) Flowchart of the protocol. (B) Membrane integrity assay sample setup in a typical 96-well format for determining optimal digitonin concentration. 6 × 103 MCF7 cells per well were seeded in a 96-well plate. (C) Plasma membrane integrity in MCF7 cells treated with digitonin in indicated concentrations, or left untreated in media with Ca2+ for 30 min. The percentage of permeabilized cells is assayed using cell impermeable PI and permeable Hoechst 33342 dye (i.e., by calculating the number of PI positive cells relative to the total number of Hoechst 33342 positive cells) for each condition. Numbers represent mean of triplicate measurements, and error bars indicate SD values. All cells were permeabilized already at 30 µg/mL digitonin. A digitonin concentration that creates plasma membrane damage that can be repaired by most cells (approximately 80%) is determined. Here, 7.5–10 µg/mL is optimal for MCF7 cells.



    Figure 4. Example of membrane integrity assay following digitonin-induced injury.

    (A) Flowchart of the protocol. (B) Membrane integrity assay sample setup in a typical 96-well format in media without and with Ca2+. (C) Plasma membrane integrity in MCF7 cells treated with 7.5 or 10 µg/mL digitonin, or left untreated in media with or without Ca2+ for 30 min. The percentage of permeabilized cells is assayed using cell impermeable PI and permeable Hoechst 33342 dyes, and calculated by relating the measurement for PI to the measurement for Hoechst positive cells for each condition. Numbers represent means of triplicate measurements, and error bars indicate SD values. (D) Representative images showing the incorporation of Hoechst 33342 and PI dyes at a single cell level in the presence and absence of Ca2+, before and after digitonin treatment (7.5 µg/mL digitonin for 30 min). Scale bar = 500 µm.

Notes

The above methods must be used with care, when using treatment that influences cellular cholesterol content, or drugs that have detergent-like properties, such as cationic amphiphilic drugs (CADs) (Petersen et al., 2013), as such treatment may interfere with the digitonin damaging ability. Protocol C describes how membrane integrity following detergent induced injury using digitonin is measured. However, this protocol can also be adjusted to study membrane integrity following toxin-induced injury, by substituting digitonin treatment with treatment with SLO, or another pore-forming toxin.

Acknowledgments

The protocols presented here were applied in our recent papers (Sønder et al., 2021; Heitmann et al., 2021; and Sønder et al., 2019). We thank both present and former colleagues from the Membrane Integrity Group, Danish Cancer Society Research Center, for optimizing and fine tuning the methods presented here. Further, we thank colleagues and collaborators within the membrane repair field for sharing their methods and knowledge, in particular Jyoti K. Jaiswal, Children’s National Research Institute. The work was supported by the Danish Council for Independent Research (6108-00378A, 9040-00252B), the Novo Nordisk Foundation (NNF18OC0034936), and the Scientific Committee Danish Cancer Society (R90-A5847-14-S2, R269-A15812).

Competing interests

The authors have nothing to disclose.

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简介

[摘要]质膜的损伤和膜完整性的丧失对真核细胞是有害的。因此,细胞必须拥有有效的膜修复系统才能生存。然而,质膜修复背后的不同细胞和分子机制尚未完全阐明。在这里,我们提出了三种互补的质膜损伤方法,以及膜修复和完整性的测量。第一个协议是基于响应激光诱导损伤的细胞膜修复动力学的实时成像。第二个和第三个协议是终点测定,提供基于群体的膜完整性测量,在涡旋与玻璃珠混合造成机械损伤后,或在亚分解浓度的洋地黄皂苷引起的洗涤剂诱导损伤后。该协议可应用于培养中的大多数贴壁真核细胞,以及悬浮细胞。


[背景] 与受细胞壁保护的原核细胞不同,真核细胞缺乏这种保护层,因此更容易受到膜损伤(Cooper 和 McNeil,2015) 。真核细胞的质膜由具有整合跨膜蛋白的磷脂双分子层组成,其本质上构成了将细胞与细胞外环境隔开的物理屏障,并维持向外部的基本渗透梯度(Khan等人,2013) 。在大多数细胞的生命周期中,质膜的完整性经常受到不同方式的损害。驻留在机械活跃的组织环境中的细胞,例如。 、肌肉和肺细胞的质膜经常受到损伤(McNeil 和 Khakee,1992 年;Gajic等人,2003 年) 。机械应力和化学应力,例如入侵病原体分泌的成孔毒素(Bischofberger等人,2009) ,都可以诱导膜损伤,如果不进行修复,就会对细胞存活构成直接威胁。因此,细胞已经开发出有效的质膜修复机制来应对膜损伤并确保细胞稳态。修复机制严格依赖于细胞外钙离子 (Ca 2+ ) 通过伤口流入细胞,并涉及多个细胞过程,包括细胞骨架重组(Abreu-Blanco et al. , 2011b) 、胞吐作用(Bi et al. , 1995; Andrews et al. , 2014) , 内吞作用(Idone et al. , 2008) , 和膜脱落(Scheffer et al. , 2014; Jimenez et al. , 2014 ; Sønder et al. , 2019) 。不同的修复机制似乎可以组合使用,具体取决于施加在膜上的损伤类型(Cooper 和 McNeil,2015;Boye 和 Nylandsted,2016) 。此外,细胞外 Ca 2+的流入触发了各种 Ca 2+激活的修复蛋白快速募集到受损膜上,包括直接参与即时修复反应的膜联蛋白家族成员,以在几秒钟内封闭孔。 AK McNeil等人,2006;Bouter等人,2011;Jaiswal等人,2014;Boye等人,2017;Sønder等人,2019) 。早期修复反应发生在损伤后的几秒到几分钟内,修复的效率和潜在机制都决定了细胞的命运:要么是细胞死亡,要么是成功的细胞修复和重组。在膜联蛋白起主要作用的初始膜重新密封后,细胞需要重组和去除受损的膜,包括胞吐和内吞事件,包括巨胞饮作用。为此,我们最近发现乳腺癌细胞使用巨胞饮作用与非经典自噬系统的成分(称为 LC3 相关的巨胞饮作用)耦合,以去除质膜的受损部分,并恢复膜完整性(Sønder等人. , 2021) .
由于各种生理应激源会导致体内细胞膜完整性的丧失,因此已经开发了几种实验方法来模拟这些条件。这些方法包括通过使细胞通过窄孔注射器、从基底上刮下附着的细胞或将细胞单层暴露于滚动的玻璃珠来限制细胞诱导损伤(PL McNeil,2001;PL McNeil等人,2001;Corrotte等人。 ,2015 年;Jaiswal等人,2014 年) 。然而,这些损伤方法很难准确复制,因为它们取决于施加在注射器上的力、刮擦前的细胞汇合度或用玻璃珠滚动,导致受伤人群的差异很大样品之间的细胞。另一方面,可以使用细菌成孔毒素获得更可控的膜损伤,例如链球菌溶血素 O (SLO),其产生大约 30 nm 的孔(Tweten,2005;Idone等人,2008) 。然而,使用成孔毒素的方法不能模拟体内机械诱导的损伤,因为这些毒素通过提取胆固醇等脂质化学改变细胞膜(Gonzalez等人,2008 年;Babiychuk等人,2011 年) 。因此,损伤类型的选择非常重要,因为它会影响对修复反应的了解。
此处介绍的第一个协议(协议 A)是一种在紫外消融激光诱导的质膜损伤后监测活细胞中细胞膜修复动力学的方法。以下两个方案(方案 B 和方案 C)是终点分析,可用于识别,例如,不同细胞条件下的修复能力缺陷。激光诱导损伤是一种非常有用的实验方法,因为它会产生局部且可控的损伤,可以与活细胞成像相结合,在修复过程中跟踪荧光标记的蛋白质(Jaiswal等人,2014 年;Sønder等人。 , 2019) 。在哺乳动物和无脊椎动物的各种研究中,激光损伤方法已用于监测细胞膜修复(McNeil等人,2003;Abreu-Blanco等人,2011a) 。
为了分析膜损伤的程度和膜修复的动力学,可以应用不同的荧光膜不可渗透染料,包括 Hoechst 33258、碘化丙啶 (PI)、FM1-43 和 FM4-64。当膜被破坏时,Hoechst 33258 和 PI 都是不透膜的,它们可以进入细胞并与核酸结合。在这里,在细胞核中产生的荧光可以量化为膜渗透性增加的量度,即膜完整性差。 FM 染料是苯乙烯基亲脂性染料,在与磷脂结合时会增加荧光强度。它们将通过膜破裂进入细胞,从而作为损伤程度和愈合反应的读数,从中可以计算修复动力学(Betz等人,1996 年;Corrotte等人,2015 年) 。然而,FM 染料与质膜结合,并且还通过内吞机制被细胞吸收,这导致在长期内与膜损伤无关的强烈细胞内染色。因此,FM 染料最适合在短期测定中测量修复动力学。对于长期检测,应使用不可渗透的 Hoechst 33258 或 PI,因为这些染料仅染色核酸,而不会像 FM 染料那样出现在内化囊泡中。对于激光诱导的损伤方案,在细胞不透性染料存在的情况下,细胞膜受到高强度单光子纳秒脉冲激光的损伤。损伤导致不渗透染料进入细胞,修复限制染料进一步进入,导致荧光平台。相反,修复失败会导致染料不断进入受损细胞,细胞内染料荧光会稳步增加。
此处介绍的最后两个协议是终点检测,并提供基于人群的监测质膜修复的措施。终点分析用于深入了解细胞和分子过程在膜修复中的参与。在研究新的修复蛋白和参与细胞修复的机制时,它们的简单性是一个优势,尤其是在同时研究多种情况时。然而,细胞修复反应是一个复杂的过程,如果没有时间分辨率就无法完全监测,这可以使用能够控制细胞膜局部损伤并允许实时监测修复反应的方法来实现。在这里介绍的两个终点测定中,膜完整性是使用膜渗透染料 Hoechst 33342 和膜不渗透染料 PI 测量的。通过使用基于显微镜的读板器( Celigo ® Imaging Cytometer),每孔测量总细胞(Hoechst 33342 阳性细胞)和透化细胞(PI 阳性细胞)的数量。使用基于图像的分析来量化膜通透性和细胞死亡的一个优点是,与酶分析相比,它提供了单细胞水平的信息。在第一个方案中,细胞通过涡旋与玻璃珠混合而受到机械损伤(方案 B),而在第二个试验中,细胞使用去污剂洋地黄皂苷(方案 C)受到化学损伤。洋地黄皂苷,一种来自洋地黄的皂苷 (Sudji et al. , 2015) ,通常在大多数实验室中用于完全裂解细胞膜。然而,它也可以在亚溶解浓度下使用,产生可以被细胞修复的质膜损伤(Boye等人,2017;Heitmann等人,2021) 。毛地黄皂苷孔形成的确切机制仍不清楚,但已知依赖于膜中的胆固醇(Sudji等,2015) 。因此,膜损伤的程度取决于质膜中的胆固醇含量和洋地黄皂苷浓度。
通过这三个协议,我们描述了不同的质膜损伤方法和膜完整性测定。我们将这些方法与感兴趣基因的 siRNA 敲低和药物治疗(例如5-(N-乙基-N-异丙基)阿米洛利 ( EIPA) 和三氟拉嗪 ( TFP)] 结合使用,以阐明不同基因的作用。修复蛋白,包括 S100A11 和膜联蛋白家族的成员,在对不同类型损伤的膜修复反应中发挥作用(Jaiswal等人, 2014;Sønder等人,2019;Boye等人,2017;Heitmann等人, 2021 年;Sønder等人,2021 年) 。

方案 A:活细胞激光损伤后的质膜修复动力学

关键字:细胞损伤, 质膜损伤, 膜损伤, 质膜修复检测, 成像, 膜完整性



材料和试剂


1. RPMI 1640,不含酚红(Life Technologies 的 Gibco ® ,通过 Thermo Fisher Scientific 购买,目录号:32404014)
2. 胰蛋白酶, TrypLE TM Express 酶(1 × ),无酚红(Life Technologies 的 Gibco ® ,通过 Thermo Fisher Scientific 购买,目录号:12604013)(储存 4°C)
3. 胎牛血清(FBS)(Life Technologies 的 Gibco ® ,通过 Thermo Fisher Scientific 购买,目录号:10270106,6%,储存 4°C)
4. GlutaMax (Life Technologies 的 Gibco ® ,通过 Thermo Fisher Scientific 购买,目录号:35050061,2 mM,储存 4°C)
5. HEPES缓冲溶液储备1 M(Sigma-Aldrich,目录号:H3375,溶于H 2 O,储存4°C)
6. 无钙成像介质(例如,汉克斯平衡盐溶液(HBSS),不含钙、镁和酚红(Life Technologies 的 Gibco ® ,通过 Thermo Fisher Scientific 购买,目录号:14175095,储存 4°C,使用预热至 37°C)
7. 在当前协议中测试的细胞
a. MCF7人乳腺癌细胞
b. HeLa人宫颈癌细胞
c. SH-SY5Y 是源自 SK-N-SH 神经母细胞瘤细胞系的三次亚克隆人细胞系
8. FM1-43(Invitrogen,目录号:T3163,1.6 µM,分装 -20°C)
9. FM4-64(Invitrogen,目录号:T13320,2.5 µM,分装 -20°C)
10. 碘化丙啶(PI)(Sigma-Aldrich,目录号:P4864,0.5 µg/mL,储备 1 mg/mL,溶于 H 2 O,储存 4°C)
11. Hoechst 33258(Sigma-Aldrich,目录号: 861405,2.5 µg/mL,4°C 储存)
12. 细胞成像介质 (CIM)(参见食谱)


设备


1. 细胞培养箱设置在37°C,5% CO 2
2. 35 毫米,1.0 号盖玻片,14 毫米玻璃底,无涂层的 MatTek培养皿( MatTek Corporation,p35G-1.0-14-C)
3. 共聚焦显微镜配备旋转圆盘、烧蚀激光器和 63 ×水物镜。在本协议中,我们使用以下显微镜和设备:
a. 倒置显微镜 Eclipse Ti -E (Nikon),带 63 ×水物镜
b. UltraVIEW VoX 旋转盘(珀金埃尔默)
c. 355 nm 紫外烧蚀激光器 (Rapp OptoElectronic )
d. 用于活细胞成像的加热室 (37 °C)


软件 


1. Volocity 软件 (PerkinElmers)
2. SysCon 软件 ( Rapp OptoElectronic ) 
3. Prism (GraphPad Software, Inc) 或 Microsoft Excel (Microsoft)


程序


A. 使用活细胞成像的激光损伤后质膜修复的实时动力学


1. 1 × 10 5 个细胞播种在 35 毫米MatTek成像培养皿中,玻璃底,并让细胞在37 °C的细胞培养箱中粘附过夜。
设置 2 - 3 道菜。根据细胞系,应调整播种细胞的数量,以在实验当天获得大约 50% 的汇合。
2. 可选步骤:用抑制剂预处理细胞,或应用 siRNA 敲低感兴趣的基因。
3. 第二天(实验当天)准备成像介质:在 37°C 下预热细胞成像介质(CIM,参见食谱)。
4. 在预热的 CIM 中制备 FM 染料溶液。
最佳染料浓度因染料和细胞系而异,应进行优化。对于 MCF7 和 HeLa 细胞,1.6 µM 的 FM1-43 效果很好。在本例中,我们在 MCF7 细胞中使用 FM1-43 染料。
注意:根据您的实验设置,也可以使用其他不可渗透的染料,例如 FM4-63、不可渗透的 Hoechst 33258 或 PI。
5. 用预热的 CIM 清洗细胞,并在盘子中加入1-2 mL 的含有 FM 染料的 CIM。
6. 将盘子放在保持在 37°C 的舞台上的显微镜支架中,并设置焦点以可视化与质膜相关的 FM 染料(图 1A;和视频 1 和 2)。我们在配备顶部培养箱的倒置显微镜 Eclipse Ti -E (Nikon) 中使用 63 ×水物镜,以保持稳定的温度。
注意:以下步骤提供了实验的一般概述。细节将根据所使用的显微镜而变化,因此用户应在进行实验之前接受其特定设置的培训。
7. 在任何完整单细胞的质膜内选择一个 1 – 2 µm 的感兴趣区域,将激光定向到该区域,并使用 355 nm 紫外消融激光照射 <4 ns,使用最佳激光功率。应调整仪器的激光功率,以获得可重复且非致命的伤害。
注:
a. 烧蚀激光总是在开始实验之前进行校准。消融激光的设置取决于仪器和细胞系,必须进行优化。在我们的实验室中,我们在系统中对 MCF7 和 HeLa 细胞使用以下设置:2.6% 功率、200 Hz 重复率、脉冲能量 >60 µJ 和脉冲长度 <4 ns。
b. 我们的系统允许制作激光条纹、矩形和斑点损伤。我们始终使用点激光损伤(激光损伤部位由图 1A 中的箭头指示,或视频中的星号),激光设置如上所示。然而,在其他系统中,例如3i激光消融系统,可以随意调整损伤区域。
c. 使用激光时,您应该始终确保激光安全,尤其是在使用脉冲激光时,因为这些激光在每个短脉冲中都可以达到非常高的功率,这使得它们非常危险。一些安全预防措施包括有一个带有激光联锁的孵化室和激光安全护目镜,当存在潜在接触眼睛的风险时应使用这些护目镜。
8. 通过在明场和荧光中每 5 秒成像一次,在受伤前开始,并在受伤后继续 5 分钟,监测修复动力学。
对于阴性对照(即无修复),使用含有 FM 染料的Ca 2+ -free CIM 重复步骤 A4-A8。对每个条件至少 10 个细胞重复步骤 A7 - A8,在三个独立实验中获得。
注意:如果检测与任何预处理相结合,研究人员应包括没有激光损伤的对照细胞样本,以排除单独预处理导致的质膜损伤。
9. 使用倒置显微镜 Eclipse Ti -E (Nikon) 与UltraVIEW配对获取图像 VoX Spinning Disk (PerkinElmer),使用 63 ×水目标。


数据分析


代表性数据
协议 A 描述了如何通过监测 FM 染料在细胞中的进入来测量激光损伤后质膜修复和活细胞后续修复的动力学。
 
1. 要量化质膜渗透性(膜完整性的读数),请使用适当的成像软件(例如Volocity )测量跨时间点的单个细胞水平的细胞内 FM 染料荧光强度。细胞内 FM 染料荧光强度是通过选择细胞内损伤部位的特定感兴趣区域(FM 染料开始进入细胞的区域:图 1A 中白色虚线勾勒的区域)来测量的。通过将每个时间点 (F) 的荧光强度标准化为受伤前的荧光强度 (F 0 ), 计算成像过程中荧光强度 (F/F 0 ) 的变化。每个条件至少应测量 10 个细胞。


图1。 响应激光损伤的细胞膜修复实时成像示例。 
( A ) 在含有 FM1-43 的培养基中,以及在存在(上图)或不存在(下图)Ca 2+的情况下,MCF7 细胞被紫外激光烧蚀损伤。受损 MCF7 细胞的代表性图像显示细胞内 FM1-43 随着时间的推移(受伤前后)的积累。白色箭头表示损伤部位,而黄色箭头表示 FM1-43 积累。白色虚线勾勒的区域说明了用于 FM1-43 荧光强度量化的区域。参见视频 1 和 2。( B )MCF7 细胞中激光损伤后单细胞膜修复动力学的量化,在有或没有 Ca 2+的介质中,测量为 FM1-43 染料荧光强度的变化 (F/F 0 )随着时间的推移,在 FM1-43 染料进入细胞的区域(损伤部位)。黑色箭头表示受伤时间点。比例尺 = 4.6 µm。


2. 荧光强度(F/F 0 :y 轴)随时间(x 轴)的平均或单个细胞变化(图 1B)。当表示为平均值时,必须考虑从三个独立实验中获得的每个条件至少 10 个细胞,并且必须绘制平均值的标准误差 (SEM)。对于统计分析,计算曲线下面积 (AUC) 分析,然后计算带有 Welch 校正的非配对 t 检验。例如更多细胞的量化,以及表示平均荧光强度的图,请参见Sønder中的图 1A 等。 (2021 年)。
3. 在受伤前的不同时间点选择每个条件的代表性图像,以组装一组图像,显示不同条件下 FM 染料的流入(有或没有 Ca 2 + )(图 1A)。 对代表性图像进行注释以演示如何选择感兴趣的区域(图 1A)。




在 MCF7 细胞中的Ca 2+介质中吸收 FM1-43 染料




视频 2 。在 MCF7 细胞中无 Ca 2+的培养基中激光损伤后 FM1-43 染料的摄取


笔记


FM 染料与脂质膜结合,但不会扩散穿过完整的质膜。然而,FM 染料也通过内吞作用被细胞吸收,使得用于测量修复动力学的测定仅适用于短期测定。进行实验的时间尺度取决于所选细胞吸收 FM 染料而没有任何损坏的速度。在我们使用 MCF7 或 HeLa 细胞的实验室中,我们通常只在添加 FM 染料后使用培养皿进行 20-30 分钟的激光损伤实验;在此之后,很难测量受伤后 FM 染料的额外流入量。
对于长期检测,应使用不渗透的 Hoechst 33258 或 PI。该测定还可以与不同的预处理相结合,例如三氟拉嗪 (TFP),或通过 siRNA 敲除感兴趣的基因。有关检测与不同预处理相结合的示例,请参见Sønder 等。 (2019),海特曼 等。 (2021)和森德 等。 (2021 年)。


食谱


1. 细胞成像介质 (CIM)
GlutaMax和 25 mM HEPES的无色 RPMI 1640 。


方案 B:使用玻璃珠涡旋混合机械损伤后的膜完整性


材料和试剂


1. 6 孔细胞培养板或 10 厘米细胞培养皿
2. 埃彭多夫管
3. 猎鹰管
4. 96 孔细胞培养板透明平底
5. 多道移液器
6. RPMI 1640(Life Technologies 的 Gibco ® ,通过 Thermo Fisher Scientific 购买,目录号: 11875085)
7. 胎牛血清(Life Technologies 的 Gibco ® ,通过 Thermo Fisher Scientific 购买,目录号:10270106)(6%,储存 4°C)
8. 汉克斯平衡盐溶液(HBSS),不含钙,不含镁,不含酚红(Life Technologies 的 Gibco ® ,通过 Thermo Fisher Scientific 购买,目录号:14175095)(储存 4 °C ,使用预热至 37°C )
9. 胰蛋白酶, TrypLE TM Express 酶(1 × ),无酚红(Life Technologies 的 Gibco ® ,通过 Thermo Fisher Scientific 购买,目录号:12604013)(储存 4°C)
10. DPBS,无钙,无镁(Life Technologies 的 Gibco ® ,通过 Thermo Fisher Scientific 购买,目录号:14190144)(储存 4°C)
11. 在当前协议中测试的细胞
a. MCF7人乳腺癌细胞
b. HeLa人宫颈癌细胞
c. MDA-MB-231 细胞,源自乳腺癌
12. 425-600 µm 玻璃珠(Sigma-Aldrich,目录号: G8772)(存储 RT)
13. bisBenzimide H 33342 trihydrochloride(以下简称 Hoechst,目录号: 33342)[Sigma-Aldrich,目录号:B2261,6.25 µg/mL,储备液(25 mg/mL),溶于 H 2 O}。 -20°C 长期保存,4°C 短期保存,可稳定保存 1 个月。
14. 碘化丙啶 (PI) [Sigma Aldrich, P4864, 0.25 µg/mL, stock (1 mg/mL), 溶于 H 2 O]。储存于4°C。


设备


1. 平衡
2. 细胞培养箱设置在37°C,5 % CO 2
3. V形水库
4. 涡流混合器
5. 定时器
6. 基于板的明场和荧光成像细胞仪,具有至少两个荧光通道(激发 377/50 和发射 477/22,以及激发 531/40 和发射 629/53),例如Celigo ®成像细胞仪(Brooks Life Science Systems )。


软件


1. Celigo 软件(布鲁克斯生命科学系统)
2. Prism (GraphPad Software, Inc) 或 Microsoft Excel (Microsoft)


程序


B. 使用玻璃珠涡旋混合机械损伤后的膜完整性


除非另有说明,否则所有细胞培养孵育均应在加湿的 37°C、5% CO 2培养箱中进行。有关协议的流程图,请参见图 2A。
1. 准备 1.5 毫升 Eppendorf 管和250 毫克玻璃珠(425 - 600 µm)。
注意:每种条件下每个细胞系至少准备三个 Eppendorf 管。
2. 根据实验,在 6 孔培养板或 10 厘米培养皿中播种适量的细胞。
注意:细胞在实验当天的汇合度不应超过 80%。
3. 可选步骤:用抑制剂预处理细胞,或对感兴趣的基因进行 siRNA 敲低。
4. 在实验当天,用胰蛋白酶消化感兴趣的贴壁细胞。
a. 从细胞培养皿中取出所有培养基。
b. 用少量不含 Ca 2+和 Mg 2+的 DPBS 清洗贴壁细胞单层一次,以去除任何可能抑制胰蛋白酶作用的残留 FBS。
注意:在不含 Ca 2+ - 和 Mg 2+ -的缓冲盐溶液中洗涤细胞,因为盐溶液中的 Ca 2+和 Mg 2+会导致细胞粘在一起。
c. 向细胞培养物中加入足够的胰蛋白酶,以覆盖贴壁细胞层。
d. 在细胞培养箱中孵育细胞 2-5 分钟。
e. 点击台面上的板底部以去除细胞。
注意:用倒置显微镜检查细胞培养物,以确保细胞被四舍五入并从表面分离。如果细胞没有充分分离,请将细胞培养物放回培养箱中再放置一两分钟。
f. 添加适当的培养基,并重新悬浮细胞。
5. 对细胞进行计数并将它们分成两支 falcon 管,一支用于含有 Ca 2+培养基中的细胞,一支用于无 Ca 2+培养基中的细胞 
注意:如果细胞已经用抑制剂或通过 siRNA 敲低感兴趣的基因进行预处理,则应对每种情况进行计数。
6. 将细胞在室温下以 300 × g离心 4 分钟,然后将细胞重新悬浮在培养基中,无论是否含有 Ca 2+ 。将 HEPES 添加到两个 falcon 管中,最终浓度为 25 mM。
7. 7 × 10 4 个细胞与 250 mg 玻璃珠 (425 – 600 µm) 一起装入1.5 mL Eppendorf 管中。
笔记:
a. 应针对不同的细胞系优化细胞数量。需要将适当数量的细胞以 350 – 400 µL 的体积装入玻璃珠。对于 MCF7 和 HeLa 细胞,我们使用 7 × 10 4细胞悬浮液和 250 mg 玻璃珠(425 – 600 µm),将 350 µL 重悬细胞以 200,000 个细胞/mL 的密度加载到玻璃珠上。
b. 作为无修复的阴性对照,包括在玻璃珠上加载 Ca 2+无介质的细胞样品。
8. 在细胞培养箱中孵育管 10 分钟。
注意:在涡旋混合损伤前,将细胞孵育以保持最佳细胞条件,包括温度 (37°C)。
9. 在 96 孔板的各个孔中添加 50 μL 的介质,无论是否带有 Ca 2+ 。
10. 通过以最大速度涡旋混合 0、30 或 60 秒来伤害细胞。
涡旋时间可能需要优化,因为某些类型的细胞比其他类型的细胞更脆弱,并且不同细胞系之间的修复效率也有所不同。最佳的涡旋时间应该在没有任何其他预处理的情况下在含有 Ca 2+的培养基中产生 10-30% 的透化细胞,因为大多数细胞需要能够在这种情况下修复它们的膜损伤。这种平衡是由于需要透化一定数量的细胞以确保细胞已被破坏,但另一方面,当细胞处于其他条件时,透化细胞仍应有增加的空间。膜修复受损的细胞被包括在测定中。
11. 受伤后立即在 96 孔板中每孔板 10,000 个细胞。 为每个条件重复三次。用手摇动板以将细胞分布在孔中。
注意:每个 96 孔应包含 50 μL 的培养基和重悬细胞(10,000 个细胞),以及在步骤 B9 中添加的 50 μL 培养基。
12. 在细胞培养箱中孵育 96 孔板 5 分钟,以便细胞修复。
13. 2+的介质中制备 PI 和 Hoechst 33342 染料混合物。
注意:最终浓度应为 0.25 µg/mL PI 和 6.25 µg/mL Hoechst 33342。
14. 小心地将 50 μL 的相应 PI/Hoechst 染料混合物添加到 96 孔板的相应孔中,并在细胞培养箱中孵育5 分钟。
15. 细胞总数(Hoechst 33342 阳性细胞:激发 350 nm,发射 461 nm)和透化细胞数(PI 阳性)确定膜完整性,使用成像细胞细胞仪(例如Celigo ® )测量膜完整性细胞:每孔激发 535 nm,发射 617 nm)。
Celigo ®是一种多通道成像细胞细胞仪,可用于使用光学显微镜进行全孔活细胞分析。在本实验中,在扫描和分析之前,用可渗透的 Hoechst 33342 染料和不可渗透的 PI 染料标记细胞。细胞仪扫描选定区域,例如选定通道中的 96 孔。然后,该软件可用于识别所选通道中的标记细胞,从而计算总细胞数(Hoechst 阳性细胞)和透化细胞数(PI 阳性细胞)。
注意:在设置实验之前,用户应该在他们的设备上接受特定的培训。


数据分析


代表性数据
协议 B 描述了如何通过涡旋与玻璃珠混合来监测机械性损伤后的膜完整性。


1. PI (透化细胞)和 Hoechst 33342(总细胞)阳性细胞的三次测量的平均值。
2. 通过将 PI 阳性细胞总数与每种条件下的 Hoechst 33342 阳性细胞总数相关联,计算每种条件下透化细胞的百分比。对于统计分析,应至少进行三个独立实验,并绘制标准偏差。有关已应用统计分析的示例,并且该测定与不同的预处理相结合,请参见Heitmann 等。 (2021)和森德 等。 (2021 年)。




图 2。 膜完整性测定的示例,在玻璃珠涡旋机械诱导损伤后。 
( A ) 协议流程图。 ( B ) 典型的 96 孔格式的膜完整性测定样品设置在含有和不含 Ca 2+的培养基中。 ( C ) MCF7 细胞悬液中的质膜完整性,在有或没有 Ca 2+的培养基中,用玻璃珠暴露于涡流 0、0.5 或 1 分钟。根据含有不可渗透 PI 的细胞数和细胞总数(通过可渗透的 Hoechst 33342 染料检测)计算透化细胞的百分比。数据代表三次测量的平均值,误差条表示 SD 值。最佳涡旋时间是在没有任何其他预处理的情况下,在含有 Ca 2+的培养基中的细胞可产生10-30 % 透化细胞的时间,因为大多数细胞需要能够在这种情况下修复其膜损伤。 在这里,1 分钟的涡旋混合是 MCF7 细胞的最佳涡旋时间,因为这会导致在与玻璃珠涡旋混合后,含有 Ca 2+的培养基中大约 12% 的通透细胞。
笔记


该测定还可以与不同的预处理组合,例如,不同生物过程的抑制剂,例如5-(N-乙基-N-异丙基)阿米洛利(EIPA),其抑制巨胞饮作用,或例如,基因的敲低siRNA 感兴趣。有关检测与不同预处理相结合的示例,请参见Heitmann 等。 (2021)和森德 等。 (2021 年)。


方案 C:使用洋地黄皂苷引起的去污剂损伤后的膜完整性
应该注意的是,洋地黄皂苷透化细胞膜的能力不仅取决于洋地黄皂苷浓度,还取决于每个细胞中洋地黄皂苷分子的总量。因此,洋地黄皂苷应始终以每个细胞相同的体积使用。


材料和试剂


1. 埃彭多夫管
2. 96 孔细胞培养板透明平底
3. 多道移液器
4. RPMI 1640(Life Technologies 的 Gibco ® ,通过 Thermo Fisher Scientific 购买,11875085)
5. 胎牛血清(Life Technologies 的 Gibco ® ,通过 Thermo Fisher Scientific 购买,目录号:10270106)(6%,储存 4 °C )
6. 在当前协议中测试的细胞
a. MCF7人乳腺癌细胞
b. HeLa人宫颈癌细胞
c. NCI-H1299 源自非小细胞肺癌
d. 源自乳腺癌的MDA-MB-231细胞
7. Digitonin (Sigma-Aldrich),Digitonin Stocks(5 和 50 mg/mL),溶于 H 2 O。储存于 -20°C。洋地黄皂苷通常会沉淀,必须通过加热和偶尔涡旋重新溶解。临用前,将洋地黄皂苷原液加热至 95°C 5 分钟,以溶解沉淀物。
8. Hoechst 33342(Sigma-Aldrich,目录号: B2261,6.25 µg/mL,储备液(25 mg/mL),溶解在 H 2 O 中。在 -20°C 下长期储存,在 4°C 下短期储存(稳定 1月)。
9. 碘化丙啶(PI)(Sigma-Aldrich,目录号:P4864,0.25 µg/mL,储备液(1 mg/mL),溶于 H 2 O。储存在 4°C。


设备


1. 细胞培养箱设置在37°C,5% CO 2
2. V形水库
3. 涡流混合器
4. 热块
5. 定时器
6. 基于平板的明场和荧光成像细胞仪,至少有两个荧光通道(激发 377/50 和发射 477/22,以及激发 531/40 和发射 629/53),例如Celigo ® Imaging Cytometer (Brooks Life科学系统)


软件


1. Celigo 软件(布鲁克斯生命科学系统)
2. Prism (GraphPad Software, Inc) 或 Microsoft Excel (Microsoft)


程序


C. 使用洋地黄皂苷引起的去污剂损伤后的膜完整性


第 1 部分:确定用于产生大多数细胞能够修复的质膜损伤的最佳洋地黄皂苷浓度(参见图 3A 的协议流程图)。
以下程序对于确定用于产生质膜损伤的最佳洋地黄皂苷浓度是必要的,这种损伤可以在含有 Ca 2+的培养基中被大多数细胞 (~80%) 修复,无需任何预处理。该程序应定期执行,因为试剂(例如,毛地黄皂苷原液)和细胞条件可能会随时间而变化。执行此程序可确保受损细胞数量的一致性以及所得结果的可重复性。该程序应分别对每个细胞系进行。


1. 在 96 孔板中的 100 µL 培养基中每孔接种6 × 10 3 个细胞,并让细胞在 37°C 的细胞培养箱中粘附过夜。制作两块板:一块板用于确定最佳洋地黄皂苷浓度,另一块板用于测量膜完整性。
调整每个细胞系的每孔细胞数,因为细胞在实验当天需要 50% 融合。
2. 立即使用前,将 digitonin 库存 (5 µg/µL) 加热至 95 °C 5 分钟以溶解任何沉淀物。
注意:洋地黄皂苷通常会沉淀,需要通过加热重新溶解,偶尔涡旋混合。
3. 在预热的培养基 ( 37 ° C ) 中制备以下十种洋地黄皂苷稀释液:0、5、7.5、10、12.5、15、20、25、30 和 100 µg/mL 洋地黄皂苷,
注意:100 µg/mL 的洋地黄皂苷溶液用于细胞的总透化/完全透化。
4. 每口井添加 100 μL 的 digitonin 稀释液,一式三份。将洋地黄皂苷溶液添加到井的一侧,以避免贴壁细胞脱离。
5. 37 °C的细胞培养箱中孵育30 分钟。同时,用 PI 和Hoechst 33342染料稀释液制备培养基;最终浓度应为:0.25 µg/mL PI 和 6.25 µg/mL Hoechst 33342。
6. 小心地将 50 μL 的 PI/Hoechst 染料混合物添加到 96 孔板中的每个孔中,并在细胞培养箱中孵育 5 分钟。
7. Celigo ®测量膜完整性 流式细胞仪,通过测量每孔的总细胞数 [Hoechst 33342 (激发 350 nm,发射 461 nm) 阳性细胞] 和透化细胞数 [PI (激发 535 nm,发射 617 nm) 阳性细胞]。
8. 确定最佳的洋地黄皂苷浓度。
a. 计算不同条件下 PI 阳性细胞的百分比。使大约 80% 的细胞能够修复 [即,大约 20% 的透化细胞(PI 阳性)] 的洋地黄皂苷浓度是最佳的。
在此示例中,MCF7 细胞用地高宁处理 30 分钟,最佳地高宁浓度为 7.5–10 µg/mL(参见图 3C)。


第 2 部分:使用洋地黄皂苷测量去污剂诱导损伤后的膜完整性
2+的培养基中用洋地黄皂苷处理的细胞的膜完整性。该测定还可以与不同的预处理相结合,例如三氟拉嗪 (TFP),或通过 siRNA 敲低感兴趣的基因。有关检测与不同预处理相结合的示例,请参见Sønder 等。 (2019)和海特曼 等。 (2021 年)。有关协议的流程图,请参见图 4A。
9. 可选步骤:用抑制剂预处理细胞。
10. 2+的介质和无 Ca 2+的介质中制备洋地黄皂苷溶液(使用在步骤 C8 中获得的最佳浓度) 。我在使用前立即将洋地黄皂苷原液 (5 µg/µL) 加热至 95 °C 5 分钟,以溶解任何沉淀物。
备注:
a. 洋地黄皂苷通常会沉淀,需要通过加热和偶尔涡旋混合来重新溶解。
b. 作为阴性对照(即无修复),包括细胞在无 Ca 2+培养基中的样品。
11. 2+的 HBSS 中洗涤细胞两次。在最后一次洗涤中,仅将不含 Ca 2+ 的 HBSS 添加到将在不含 Ca 2+的培养基中用洋地黄皂苷处理的孔中。其余的孔应包含正常的培养基。
12. 如步骤 C4 中所述,每口井添加 100 μL 的每个 digitonin 稀释液,一式三份。 将洋地黄皂苷溶液添加到井的一侧,以避免贴壁细胞脱离。
13. 37 °C的细胞培养箱中孵育30 分钟。同时,在含有和不含 Ca 2+的介质中制备PI 和 Hoechst 33342 染料稀释液。最终浓度应为 0.25 µg/mL PI 和 6.25 µg/mL Hoechst 33342。
14. 小心地将 50 μL 的相应 PI/Hoechst 染料混合物添加到 96 孔板的相应孔中,并在细胞培养箱中孵育 5 分钟。
15. 通过使用总细胞数( Hoechst 33342阳性细胞:激发 350 nm,发射461 nm )和透化细胞数(PI 阳性细胞:每孔激发 535 nm,发射 617 nm)。
Celigo ®是一种多通道成像细胞细胞仪,可用于使用光学显微镜进行全孔活细胞分析。在本实验中,在扫描和分析之前,用可渗透的 Hoechst 33342 染料和不可渗透的 PI 染料标记细胞。细胞仪扫描选定区域,例如选定通道中的 96 孔。然后,该软件可用于识别所选通道中的标记细胞,从而计算总细胞数(Hoechst 阳性细胞)和透化细胞数(PI 阳性细胞)。
注意:在设置实验之前,用户应该在他们的设备上接受特定的培训。


数据分析


代表性数据
协议 C 描述了如何在洗涤剂洋地黄皂苷引起化学损伤后监测细胞膜的完整性。首先,确定产生可由细胞修复的质膜损伤的最佳洋地黄皂苷浓度。


1. 计算每个条件下 PI(透化细胞)和 Hoechst(总细胞)阳性细胞的三次测量的平均值。
2. 通过将 PI 阳性细胞总数与 Hoechst 阳性细胞总数相关联,计算每种条件下透化细胞的百分比。对于统计分析,应至少进行三个独立实验,并绘制标准偏差。有关已应用统计分析以及将测定与预处理相结合的示例,请参见Heitmann 等。 (2021 年)。
3. 2+的情况下,在毛地黄皂苷处理之前和之后,Hoechst 33342 和 PI 染料在单个细胞水平上的结合(图 4D)。




图 3。 优化实验示例,用于确定使用 MCF7 细胞进行膜完整性测定的最佳洋地黄皂苷浓度。 
( A ) 协议流程图。 ( B ) 典型的 96 孔格式的膜完整性测定样品设置,用于确定最佳的洋地黄皂苷浓度。将每孔6 × 10 3 个MCF7 细胞接种在 96 孔板中。 ( C ) 用指定浓度的洋地黄皂苷处理的 MCF7 细胞中的质膜完整性,或在含有 Ca 2+的培养基中未处理 30 分钟。对于每种条件,使用细胞不可渗透的PI和可渗透的Hoechst 33342染料(即,通过计算PI阳性细胞相对于Hoechst 33342阳性细胞总数)来测定透化细胞的百分比。数字代表三次测量的平均值,误差条表示 SD 值。所有细胞均已在30 µg/mL 洋地黄皂苷下透化。确定了可被大多数细胞修复的质膜损伤(约 80%)的洋地黄皂苷浓度。在这里,7.5 – 10 µg/mL 是 MCF7 细胞的最佳浓度。






图 4。 洋地黄皂苷诱导损伤后膜完整性测定的示例。 
( A ) 协议流程图。 ( B ) 典型 96 孔格式的膜完整性测定样品设置在不含和含 Ca 2+的培养基中。 ( C ) 用 7.5 或 10 µg/mL 洋地黄皂苷处理的 MCF7 细胞中的质膜完整性,或在有或没有 Ca 2+的培养基中未处理 30 分钟。使用细胞不可渗透的 PI 和可渗透的 Hoechst 33342 染料测定透化细胞的百分比,并通过将 PI 的测量值与每种条件下的 Hoechst 阳性细胞的测量值相关联来计算。数字代表三次测量的平均值,误差条表示 SD 值。 (D) 具有代表性的图像,显示在存在和不存在 Ca 2+的情况下,Hoechst 33342 和 PI 染料在单细胞水平上的掺入,在地高辛处理前后(7.5 µg/mL 地高辛处理 30 分钟)。比例尺 = 500 µm。


笔记


在使用影响细胞胆固醇含量的治疗或具有类似去污剂特性的药物(如阳离子两亲性药物 (CAD))时,必须小心使用上述方法(Petersen等人,2013 年) ,因为此类治疗可能会干扰洋地黄皂苷的破坏能力。协议 C 描述了如何测量使用洋地黄皂苷引起的洗涤剂损伤后的膜完整性。然而,该协议也可以调整以研究毒素引起的损伤后的膜完整性,方法是用 SLO 或另一种成孔毒素治疗代替地黄皂苷治疗。


致谢


这里介绍的协议在我们最近的论文中得到应用( Sønder 等人,2021;海特曼 等人,2021;和森德 等人,2019)。我们感谢丹麦癌症协会研究中心膜完整性小组的现任和前任同事优化和微调这里介绍的方法。此外,我们感谢膜修复领域的同事和合作者分享他们的方法和知识,特别是儿童国家研究所的 Jyoti K. Jaiswal。这项工作得到了丹麦独立研究委员会 (6108-00378A、9040-00252B)、诺和诺德基金会 (NNF18OC0034936) 和丹麦癌症协会科学委员会 (R90-A5847-14-S2、R269-A15812) 的支持。


利益争夺


作者没有什么可透露的。


参考


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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Sønder, S. L., Ebstrup, M. L., Dias, C., Heitmann, A. S. B. and Nylandsted, J. (2022). Plasma Membrane Wounding and Repair Assays for Eukaryotic Cells. Bio-protocol 12(11): e4437. DOI: 10.21769/BioProtoc.4437.
  2. Sønder, S. L., Häger, S. C., Heitmann, A. S. B., Frankel, L. B., Dias, C., Simonsen, A. C. and Nylandsted, J. (2021). Restructuring of the plasma membrane upon damage by LC3-associated macropinocytosis. Sci Adv 7(27): eabg1969
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