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Using xCELLigence RTCA Instrument to Measure Cell Adhesion
使用xCELLigence RTCA仪器测量细胞粘附   

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The Journal of Cell Biology
Apr 2017

Abstract

Cell adhesion to neighbouring cells and to the underlying extracellular matrix (ECM) is a fundamental requirement for the existence of multicellular organisms. As such, the formation, stability and dissociation of cell adhesions are subject to tight control in space and time and perturbations within the sophisticated adhesion machinery are associated with a variety of human pathologies. Here, we outline a simple protocol to monitor alterations in cell adhesion to the ECM, for example, following genetic manipulations or overexpression of a protein of interest or in response to drug treatment, using the xCELLigence real-time cell analysis (RTCA) system.

Keywords: xCELLigence (xCELLigence), Real-time cell analysis (实时细胞分析), Cell adhesion (细胞粘附), Extracellular matrix (细胞外基质), Integrins (整合素)

Background

The principal molecules responsible for cell adhesion to the underlying ECM are a family of transmembrane heterodimeric receptors named integrins. Integrin activation and binding to the ECM triggers the recruitment of a vast array of signalling, scaffolding and cytoskeletal proteins to the integrin cytoplasmic tails. Together, these adhesion constituents represent a complex and highly dynamic machinery responsible for regulating many important cellular processes including cell proliferation, survival, migration and differentiation. In line with significant roles in maintaining normal physiological functions, dysregulated integrin-mediated adhesion and signalling is a forerunner in the pathogenesis of many human ailments, including bleeding disorders, cardiovascular disease and cancer (Giancotti and Ruoslahti, 1999; Bökel and Brown, 2002; Huveneers and Danen, 2009; Legate et al., 2009; Bouvard et al., 2013; Calderwood et al., 2013; Horton et al., 2015; Seguin et al., 2015). Therefore, investigation of integrin-dependent cell-ECM adhesions is an intensely studied research topic and of broad interest to many biological fields.

In addition to a variety of other techniques, we (Georgiadou et al., 2017; Lilja et al., 2017; Närvä et al., 2017) and others (Kiely et al., 2015; Böhm et al., 2016; Salmela et al., 2016) have used the xCELLigence RTCA system as a simple, yet quantitative, method to monitor changes in cell-ECM adhesion. This technology works by measuring electron flow transmitted between gold microelectrodes, fused to the bottom surface of a microtiter plate, in the presence of an electrically conductive solution such as tissue culture medium (Figure 1). Adhering cells disrupt the interaction between the electrodes and the bulk solution and thus impede electron flow. This impedance (resistance to alternating current) is expressed as arbitrary units called cell index (Figure 1), the magnitude of which is dependent on cell number, cell morphology and cell size and on the strength of cell attachment to the substrate coating the plate. The advantage of using xCELLigence RTCA, as opposed to traditional dye- or microscopy-based analysis of cell adhesion (Chen et al., 2009; Humphries, 2009; Humphries et al., 2009; Chen, 2012), is that a continuous readout, rather than a single time-point or end-point analysis, of cell adhesion can be obtained from the moment the cells begin to attach to the substrate. Moreover, measurements are based on the whole cell population rather than individually selected cells and therefore results are less likely to be subject to bias. However, there are situations where analysing adhesion based on a cell population is flawed. For example, when the effects of protein overexpression or knockdown are being investigated and the transfection efficiency is extremely low, the xCELLigence system will not provide a true reflection of the experimental manipulation on cell adhesion. Indeed, in these cases microscopy based imaging of known adhesion components, to monitor changes in the size and/or morphology of cell-ECM contacts, and the actin cytoskeleton, to monitor cell size and cell spreading, in individual transfected cells would be a more suitable approach. Nevertheless, the inclusion of a 96-well E-plate format within the xCELLigence RTCA single plate (SP) model enables testing of multiple experimental conditions at the same time and in the same plate, thus reducing experimental variability and allowing quick deduction of optimal assay parameters (e.g., ECM ligand concentration or time-point/s of adhesion) for more comprehensive analyses that may require more costly reagents and additional optimisation.

We have in the past used xCELLigence RTCA to analyse cell adhesion in MDA-MB-231 (triple-negative human breast adenocarcinoma) cells and HEK293 (human embryonic kidney) cells (Lilja et al., 2017), in MEFs (mouse embryonic fibroblasts) (Georgiadou et al., 2017) and in human iPSCs (induced pluripotent stem cells) (Närvä et al., 2017). We found that loss of the metabolic regulator AMPK (Georgiadou et al., 2017) or the postsynaptic density scaffolding protein Shank (Lilja et al., 2017) promotes cell adhesion to ECM molecules over time, supporting a role for these two proteins as novel inhibitors of integrin function. We have also used xCELLigence to demonstrate the different adhesive properties of human iPSCs following differentiation (Närvä et al., 2017). Here, we will describe the xCELLigence RTCA cell adhesion protocol for HEK293 cells (Lilja et al., 2017) and indicate, where appropriate, the optimisation steps required for other cell types.


Figure 1. Using the xCELLigence RTCA system to monitor cell adhesion. A. A simplified cartoon of an E-plate 96 and the gold microelectrodes embedded within each well (top view of a single well) is shown on the left. The number and size of the gold microelectrodes are not representative of the actual set-up and are for illustrative purposes only. The E-plate 96 is placed within the RTCA SP Station (1), which is kept in a humidified incubator and is connected to the RTCA Analyzer (2) and RTCA Control Unit (3). B. Workflow of an xCELLigence adhesion assay. Here, the example assay sets out to determine the optimal concentration of two different ECM ligands (purple and green) needed to promote efficient cell adhesion and to be used for further analyses. 1. The wells in the E-plate 96 are coated with serial dilutions of the ECM ligands or with BSA (yellow) as a negative control. All conditions are performed in triplicate. 2. Coating solutions are removed, wells are washed with PBS and blocked with 0.1% BSA (yellow tube) to prevent non-specific cell adhesion and then incubated with the base medium (pink) that is specific for the cell line of interest. 3. The plate is then placed in the RTCA SP station in the incubator and a reading is taken. Here, a side view of a single well in the E-plate demonstrates unimpeded electron flow from the negative to the positive terminal in the presence of medium alone, which results in a low background reading. 4. Cells are then added to the coated wells (Note: Some wells can be kept cell-free (medium-only wells) as another negative control that should only give background readings). 5. The plate is then placed back into the xCELLigence system and cell adhesion is monitored over time. Here, a side view of a single well in the E-plate demonstrates impeded electron flow from the negative to the positive terminal in the presence of adhering cells, which results in increasing impedance over time as more cells adhere and spread on the microelectrodes. *Impedance in electron flow (resistance to an alternating current) is plotted as arbitrary units called cell index. Background readings: obtained from medium alone (pink line) or from BSA-coated (yellow line) wells. Experimental readings: for simplicity, cell adhesion is shown for one of the ECM ligands (dark to light purple; high to low concentration of ligand) and for one technical replicate only. In this graphical illustration, two concentrations of the ECM ligand resulted in overlapping curves and similarly high cell index values. Therefore, the lower of these two concentrations could be used in subsequent experiments to preserve on material and reduce costs.

Materials and Reagents

  1. Pipette tips
  2. Tissue culture treated dishes (CELLSTAR® 100 x 20 mm, Greiner Bio One International, catalog number: 664160 ; 6-well, Greiner Bio One International, catalog number: 657160 ; 96-well, Greiner Bio One International, catalog number: 655160 )
  3. Falcon 15-ml conical centrifuge tubes (Corning, Falcon®, catalog number: 352196 )
  4. Falcon 50-ml conical centrifuge tubes (Corning, Falcon®, catalog number: 352070 )
  5. Microcentrifuge tubes, 1.5 ml (SARSTEDT, catalog number: 72.690.001 )
  6. Minisart® 0.45 µm single-use filters (Sartorius, catalog number: 16537-K )
  7. 60 ml syringes (BD, catalog number: 300866 )
  8. 96-well E-plates (E-plate 96) (ACEA Bio, catalog number: 5232368001 )
  9. Cell line of interest, e.g., HEK293 cells (ATCC, catalog number: CRL-1573 )
  10. Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: D1408 )
  11. ECM molecule of choice e.g., fibronectin (bovine plasma) (Sigma-Aldrich, catalog number: 341631 ); collagen (collagen from calf skin) (Sigma-Aldrich, catalog number: C8919 )
  12. HyCloneTM HyQTase cell detachment reagent (GE Healthcare, HyCloneTM, catalog number: SV30030.01 )
  13. Dulbecco’s modified Eagle’s medium (DMEM) with high glucose (4,500 mg/L) (Sigma-Aldrich, catalog number: D5671 )
  14. L-Glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030149 )
  15. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F7524 )
  16. Trypsin-EDTA for cell culture (Sigma-Aldrich, catalog number: T4049 )
  17. ≥ 96% pure bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A8022 )
  18. Appropriate growth medium for culturing cell line of interest (see Recipes)
  19. Appropriate base medium to be used in experiment (see Recipes)
  20. Fibronectin-collagen mix (see Recipes)
  21. 0.1% BSA (see Recipes)

Equipment

  1. Adjustable-volume pipettes (e.g., Fisher Scientific, model: FisherbrandTM EliteTM )
  2. Water bath
  3. Multichannel pipettes (e.g., Fisher Scientific, model: FisherbrandTM EliteTM )
  4. 37 °C, 5% CO2 water jacketed incubator (e.g., Thermo Fisher Scientific, Thermo ScientificTM, model: Series 8000 Water-Jacketed CO2 Incubators )
  5. Cell culture laminar hood (e.g., NuAire CellGardTM)
  6. Tabletop centrifuge for 15 ml and 50 ml conical tubes (e.g., Eppendorf, model: 5804 )
  7. Bürker cell-counting chamber (e.g., BRAND, catalog number: 719520 )
  8. Bright-field microscope (e.g., Olympus, model: CKX41 or ZEISS Axio Vert)
  9. xCELLigence RTCA SP Instrument (ACEA Bio, catalog number for complete system: 00380601030 ) which consists of:
    1. RTCA Analyzer (ACEA Bio, model W830, catalog number: 05228972001 )–an electronic analyser that measures, processes and analyses the impedance detected by sensor electrodes
    2. RTCA SP Station (ACEA Bio, catalog number: 05229057001 )–E-plate holder that is placed inside the incubator and connects the E-plate to the RTCA Analyzer
    3. RTCA Control Unit (ACEA Bio, catalog number: 05454417001 )–laptop with pre-installed RTCA Software

Software

  1. RTCA Software (version number 1.2.1.1002)
  2. Microsoft Excel
  3. GraphPad Prism 6 (version 6.05)

Procedure

Notes:

  1. Perform all steps under sterile conditions.
  2. Avoid touching the underside of the plate where the detectors are located.
  3. Avoid scratching the electrodes located at the bottom of the well. This can occur by pipette tips coming into contact with the electrodes. When removing solutions, tilt the plate slightly and place pipette at the side of the well and gently pipette the solution up.
  4. Pre-warm HyQTase (for detaching cells) and base medium (see Recipes) at 37 °C in a water bath prior to the experiment.
  5. Use at least 3-4 wells as technical replicates for each condition being tested.
  6. Avoid errors in cell counting and/or cell clumping in the E-plate 96 wells by thoroughly mixing the cell suspension and ensuring sufficient cell-cell dissociation prior to counting/seeding. Use multichannel pipettes, where appropriate, to ensure even addition of cells to each well.
  7. Optimise the number of cells plated for every cell line being tested; for monitoring cell adhesion to the ECM, it is best to avoid overcrowding of cells/confluent monolayers.
  8. The maximum recommended volume for each well of an E-plate 96 is 200 μl.
  9. Refer to the troubleshooting guide (see Table 1 in Notes section) for more information.

  1. Preparing the E-plates
    1. Wash wells with 150 μl sterile PBS and then aspirate the buffer.
    2. Coat wells with 100 μl ECM molecule of choice or with 100 μl 0.1% BSA (negative control, see Recipes) at 37 °C for 1 h.
      Note: The choice of ECM coating will depend on the cells being used. For HEK293 cells, which are weakly adhering cells, we have in the past used a mix of fibronectin and collagen ECM molecules (see Recipes).
    3. Remove coating and wash twice with 150 μl PBS.
    4. Block non-specific cell binding to the E-plate by incubating all wells with 100 μl 0.1% BSA at 37 °C for 1 h.
    5. Remove BSA and wash twice with 150 μl PBS.
    6. Add 50 μl of pre-warmed base medium (see Recipes) into each well and leave the E-plate in the incubator (37 °C), for at least 15 min before starting the experiment, to ensure that the culture medium and E-plate surface achieve equilibrium.

  2. Setting up the xCELLigence RTCA program
    Note: Program set-up needs to be completed prior to starting the experiment.
    1. Start the RTCA program.
    2. Set up the Exp Notes page (Figure 2A)–select File Directory in which to save the experiment files, fill in the experimental information, such as an experiment date, cell lines and treatments, an experimental procedure, a purpose and any additional information you would like to be saved.
    3. Set up the Layout page (Figure 2B). This page records the experiment layout for the run. Note that unmarked wells will not be scanned. Select a single well or multiple wells (replicates) at once (selected wells will be highlighted). Enter appropriate information for each well, such as cell type, number of cells, used compounds, etc. For example: Cell Type–HEK293, control, Cell number–20,000.


      Figure 2. Setting up the xCELLigence RTCA program–experimental details. An example of the Exp Notes (A) and the Layout page (B) for an experiment performed with HEK293 cells is shown. Four technical replicates are included for each condition. Details of the selected well in blue can be seen at the top of the page.

    4. Set up the Schedule page (Figure 3) for the plate running procedure. Experiments can be divided into multiple Steps which consist of one or several sweeps. One sweep consists of one scan across all selected wells (one measurement per well).
      1. Step 1 is considered to be the background (baseline) measurement, i.e., a scan of the E-plate prior to addition of cells (wells contain base-medium only). Step 1 is preprogrammed to be one sweep. Do not change the settings for Step 1.
      2. Make a separate Step for each time an E-plate needs to be removed from the RTCA instrument using the Add a Step option (Figure 3) (for example, add Step 2 for cell addition and Step 3 for compound addition).
      3. For continuous scanning, without pause, but with different intervals between sweeps, e.g., if measuring events occurring at different rates, use Add a Substep and edit the Interval and Sweeps by entering the appropriate values and click Apply. In the example schedule below the program will pause after Step 1 but will automatically move from Step 2.1 (substep) to Step 2.2 (substep) without the need for manual confirmation. Shorter intervals between sweeps are useful for detecting quick and potentially short-lived events, e.g., initial cell attachment to the substrate.
        An example set up for HEK293 cells:
        1. Step 1 (background measurement); 1 sweep (total time 00:00:06).
        2. Step 2.1 (e.g., substep to monitor initial cell attachment to substrate); 16 sweeps, 2 min interval (total time 00:30:06).
        3. Step 2.2 (e.g., substep to monitor subsequent cell spreading and adhesion to substrate); 18 sweeps, 5 min interval (total time 02:00:06).
        An example set up for MEFs:
        1. Step 1 (background measurement); 1 sweep (total time 00:00:06).
        2. Step 2 (to monitor cell attachment and spreading); 18 sweeps, 10 min interval (total time 03:00:06).
        Note: It is important to optimise the number of sweeps and intervals for your cell line of interest. To avoid wasting E-plates, first use normal 96-well plates to discover the timeline of adhesion for your cells, i.e., while some cells adhere very quickly (few minutes) and spread within 2 h, other cells may take longer to adhere and spread (3-4 h). When performing the xCELLigence adhesion experiment for the first time, you can use short intervals (e.g., as in Step 2.1 above) for 1 h to monitor cell index and then adjust the parameters for the next experiment accordingly.


        Figure 3. Setting up the xCELLigence RTCA program schedule–Steps and Sweeps. An example of a Schedule page is shown for HEK293 cells. Step 1 (A) is preprogrammed and should not be changed. Use Add a Step and/or Add a Substep (A) to insert additional experimental steps with different sweeps and intervals in the schedule (B).

  3. Performing the cell adhesion experiment using xCELLigence RTCA–background reading
    1. Once the program has been set up (see above), place the pre-prepared and pre-warmed E-plate containing 50 μl of base medium into the RTCA SP Station.
    2. Click Start to begin the experiment. This will initiate Step 1 for measuring the background impedance of the base medium. This reading is used as reference impedance for cell index values.
    3. A Step-Status will now display TEST. Check the Message page to see if the status and connections of the wells measured are OK.
      Important: Exclude any wells that give abnormal readings. Baseline readings are expected to be close to zero. Refer to troubleshooting section (Table 1).
    4. After completion of Step 1, the Step-Status displays DONE and the program is ready for the next step.
      Note: The RTCA program does not move onto the next Step until it is manually instructed to do so.
    5. Remove the E-plate from the RTCA SP Station and move on to adding cells.

  4. Performing the cell adhesion experiment using xCELLigence RTCA–adding cells
    1. Remove the growth medium from HEK293 cells (see Recipes). Wash cells with 5 ml of sterile PBS.
    2. Remove PBS, add 2 ml of pre-warmed HyQtase (volumes are adjusted for a confluent 10 cm culture dish) and incubate plates at 37 °C until all the cells have detached.
    3. Add 5 ml of base medium and then collect the cells by centrifugation (180 x g, 4 min).
      Note: If you use trypsin to detach cells, it is advised to subsequently inhibit trypsin activity either by using trypsin inhibitors or by adding 5 ml growth medium containing serum. We recommend using HyQtase, or other similar products, rather than digestive enzymes such as trypsin to detach cells for short-term assays where integrin function is under investigation. Integrins are sensitive to trypsin cleavage and therefore use of trypsin can affect the time taken for cells to adhere to the coated E-plates. In some cases, when cells are extremely adherent, the use of trypsin may be unavoidable and required for ECM degradation to break the cell-ECM linkage.
    4. Discard the supernatant and re-suspend the remaining cell pellet in 4 ml of base medium.
      Important: Pipet up and down gently multiple times to break cell clumps.
      Note: If using growth medium to inhibit trypsin, ensure the medium is removed completely following centrifugation, re-wash the pellet with serum-free base medium and repeat the centrifugation step above.
    5. Count cells using a method of choice (e.g., Bürker cell counting chamber), prepare a cell suspension of 2 x 105 cells/ml (dilute in base medium) and transfer 100 μl (20,000 cells; optimised for the HEK293 cell line) to each well of the pre-prepared E-plate (already contains 50 μl of base medium; therefore the total volume in each well is now 150 μl).
      Note: Cell numbers must be optimised for each cell line of interest so that the cells have enough space to adhere and spread. It is advised to use normal tissue culture treated 96-well plates and a bright-field microscope for this optimisation as the E-plate 96 are more costly. We have in the past used the following cells and cell numbers in xCELLigence cell adhesion assays: MEFs and human telomerase-immortalised fibroblasts: 7,500-10,000; MDA-MB-231 cells: 15,000-20,000; human iPSCs: 50,000. However, these are guidelines only. Different experimental treatments or culture conditions may impact on cell survival or on cell size and it is best to optimise the number of cells accordingly.
      Important: When performing a large number of conditions, it is possible that the order in which cells are added to the wells may affect the data. For example, if control cells adhere very quickly, and you expect that treated cells will respond more slowly, this difference may be exaggerated if control cells are always added first to the wells and several minutes have passed before treated cells can be plated. To overcome this potential problem, try using multichannel pipettes whenever possible to speed up cell addition and try changing the order in which cells are added to the wells in biological repeats to see if data are being influenced by the order of cell addition. If this seems to be the case, consider reducing the complexity of the experiment to reduce the intervals between cell plating.
    6. Reinsert the E-plate into the RTCA SP Station and click Start to scan Step 2.
      Important: The first measurement of Step 2 should be considered as time point 0 when analysing the data.
    7. During the experiment, you can follow the Plot page to see the cell index in real time. To do this, select the well/wells of interest and click Add. By clicking Add All every well included in the experiment is plotted.
    8. After completion of the experiment, open the Plot page, click Add All and select a full-time scale if you wish to collect the data from the entire experiment. If you click Average, the replicate wells (wells with the same name) will be combined and only the average cell index values are exported. Export raw data (cell index versus time) into an Excel spreadsheet by selecting Plate–Export Experiment Info.

Data analysis

  1. For experimental design, to compare two data sets, we perform the xCELLigence adhesion assay a minimum of three independent times. For each experiment, we use a minimum of three technical replicates.
  2. Cell adhesion is presented as changes in mean cell index values in real-time (see graphical illustration in Figure 4). We typically observe standard deviations (SDs) in the range of 0.001-0.15 for technical replicates and 0.02-0.2 for n = 4 biological replicates (the highest SD occurs at the later time points). We perform statistical analysis between the end-point cell index values of the two data sets using GraphPad Prism and the Student’s t-test.


    Figure 4. Analysis of xCELLigence cell adhesion results from two data sets. An example analysis of xCELLigence RTCA data shows cell adhesion as mean cell index ± SEM against time. Here, time point 0 denotes the point at which the E-plate 96 was first scanned after cell addition. Initial scanning of the E-plate 96 (Step 1) to monitor background readings in the absence of cells is not included in the analysis. Statistical analyses are performed for the end-point cell index values between two datasets (highlighted by a light purple box). 

Notes

  1. The cell adhesion assay is relatively simple and reproducible. However, one common problem associated with this assay is that cells may accumulate in the middle of the well rather than across the whole well after seeding. This will result in inaccurate cell index values and increased variability between experiments. In the xCELLigence manual, it is recommended to keep the plate at RT for 30 min after cell seeding to allow cells to settle to the bottom of the plate; however, this is not an option for the cell adhesion assay as cells will begin to adhere immediately and thus records of these early adhesion events would be lost. Therefore, it is vital that the plate is returned to the RTCA SP Station E-plate holder as soon as possible after cell plating and that Step 2 is started immediately. As outlined in our protocol, we recommend pre-warming the E-plate and medium and thorough mixing of cells before plating and after plating using a multichannel pipette if possible. Additionally, once cells have been seeded, we have found that gentle agitation of the plate on a surface in a clockwise followed by an anti-clockwise direction helps to disperse the cells more evenly across the well. Nevertheless, if performing the assay for the first time or if using a new cell line, in particular epithelial or endothelial cells, which prefer to have cell-cell contacts, we recommend using a normal tissue-culture treated 96-well plate to optimise cell numbers and the technique for cell addition.
  2. The E-plates used here are not suitable for bright-field imaging. Although specialised E-plates can be purchased for the purpose of obtaining phase-contrast images, these are more costly. Moreover, it is not advisable to stop the RTCA program or to remove the E-plate for prolonged periods except for predefined steps such as drug addition. If a visual inspection of the cells is desired, we recommend using a normal tissue-culture treated 96-well plate, set up in exactly the same way (i.e., same coating, blocking and number of cells) and running in parallel to the xCELLigence adhesion assay.
  3. Troubleshooting guide (Table 1)

    Table 1. Troubleshooting guide for the xCELLigence cell adhesion assay. This table contains some advice for common problems that the user may encounter during the assay. However, this table is not exhaustive and users should refer to the manufacturer’s manual for further advice if necessary.

Recipes

  1. HEK293 growth medium
    445 ml sterile Dulbecco’s modified Eagle’s medium (DMEM) with high glucose
    5 ml sterile 200 mM L-glutamine
    50 ml sterile fetal bovine serum (FBS)
    Store at 4 °C for up to 1 month
  2. HEK293 base medium (growth medium without serum)
    495 ml sterile DMEM
    5 ml sterile 200 mM L-glutamine
    Store at 4 °C for up to 1 month
  3. ECM molecule of choice
    Fibronectin-collagen mix:
    Prepare a mix of 10 μg/ml fibronectin and 20 μg/ml collagen by diluting sterile stock solutions in sterile PBS
    Important: Do not filter the working solution as this can lead to the loss of the ECM molecule on the filter. Use only freshly prepared working solutions.
    Note: For other cell lines with different adhesive properties, and if using other ECM proteins, it is advised to first test the optimal ECM ligand concentration for your specific experiment (Figure 1).
  4. 0.1% BSA
    Dilute 0.1 g of 96% BSA in 100 ml of sterile PBS and filter sterilise
    Use only freshly prepared working solutions

Acknowledgments

This work was supported by grants from the Academy of Finland, ERC Consolidator Grant (615258), the Sigrid Juselius Foundation and the Finnish Cancer Organization. J.L. is supported by Turku Doctoral Programme of Molecular Medicine (TuDMM). This protocol was adapted from procedures published in Georgiadou et al. (2017), Lilja et al. (2017), Närvä et al. (2017). The authors declare no conflicts of interest.

References

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  18. Seguin, L., Desgrosellier, J. S., Weis, S. M. and Cheresh, D. A. (2015). Integrins and cancer: regulators of cancer stemness, metastasis, and drug resistance. Trends Cell Biol 25(4): 234-240.

简介

细胞与相邻细胞和基础细胞外基质(ECM)的粘附是多细胞生物存在的基本要求。 因此,细胞粘附的形成,稳定和解离在时间和空间上受到严格的控制,复杂的粘附机制内的干扰与各种人类病理有关。 在这里,我们概述了一个简单的协议,以监测细胞粘附到ECM的变化,例如,在遗传操作或目标蛋白的过表达或响应药物治疗后,使用xCELLigence实时细胞分析(RTCA)系统。

【背景】负责细胞粘附到下面的ECM的主要分子是称为整联蛋白的跨膜异二聚体受体家族。整合素的激活和与ECM的结合触发向整合素胞质尾部募集大量信号传导,支架和细胞骨架蛋白。总之,这些粘附成分代表了负责调节许多重要细胞过程(包括细胞增殖,存活,迁移和分化)的复杂且高度动态的机制。与维持正常生理功能的重要角色一致,整合素介导的粘附和信号传导失调是许多人类疾病(包括出血性疾病,心血管疾病和癌症)发病的先导(Giancotti and Ruoslahti,1999;Bökeland Brown,2002; Huveneers and Danen,2009; Legate等人,2009; Bouvard等人,2013; Calderwood等人,2013; Horton等人,等,2015; Seguin等,,2015)。因此,整合素依赖性细胞 - 细胞外基质粘附的研究是一个研究热点,也是许多生物学领域广泛关注的课题。

除了其他多种技术之外,我们(Georgiadou等人,2017; Lilja等人,2017;Närvä等人, ,2017)和其他人(Kiely等人,2015;Böhm等人,2016; Salmela等人,2016)已经使用xCELLigence RTCA系统作为监测细胞ECM粘附的变化的简单而定量的方法。该技术通过测量在导电溶液如组织培养基(图1)存在下融合到微量滴定板底部表面的金微电极之间传输的电子流来工作。粘附的细胞破坏了电极与本体溶液之间的相互作用,从而阻碍电子流动。这种阻抗(抗交流电)表示为称为细胞指数的任意单位(图1),其大小取决于细胞数量,细胞形态和细胞大小,以及细胞附着于板上基质的强度。与传统的基于染料或显微镜的细胞粘附分析相反,使用xCELLigence RTCA的优势(Chen等人,2009; Humphries,2009; Humphries等人 >,2009; Chen,2012)是从细胞开始附着到基质的时刻开始,可以获得细胞粘附的连续读数,而不是单个时间点或终点分析。此外,测量是基于整个细胞群而不是单独选择的细胞,因此结果不太可能受到偏差。但是,有分析基于细胞群的粘附性有缺陷的情况。例如,当研究蛋白质过表达或敲低的影响并且转染效率非常低时,xCELLigence系统将不能真实反映对细胞粘附的实验操作。事实上,在这些情况下,在个体转染的细胞中,基于显微镜的已知粘附成分的成像,以监测细胞-ECM接触的大小和/或形态学的变化,以及肌动蛋白细胞骨架,以监测细胞大小和细胞扩散,将是更多合适的方法。尽管如此,在xCELLigence RTCA单板(SP)模型中包含96孔E形板格式,可以同时在同一个平板上同时测试多个实验条件,从而降低了实验的可变性,并可以快速推导出最佳分析方法参数(例如,ECM配体浓度或时间点/粘附时间)进行更全面的分析,这可能需要更昂贵的试剂和额外的优化。
负责细胞粘附到下面的ECM的主要分子是称为整联蛋白的跨膜异二聚体受体家族。整合素的激活和与ECM的结合触发向整合素胞质尾部募集大量信号传导,支架和细胞骨架蛋白。总之,这些粘附成分代表了负责调节许多重要细胞过程(包括细胞增殖,存活,迁移和分化)的复杂且高度动态的机制。与维持正常生理功能的重要角色一致,整合素介导的粘附和信号传导失调是许多人类疾病(包括出血性疾病,心血管疾病和癌症)发病的先导(Giancotti and Ruoslahti,1999;BökelandBrown,2002; Huveneers和Danen,2009;使节等人,2009年;布瓦尔等人,2013;考尔德伍德等人,2013;霍顿等人,等,2015年;因此,整合素依赖性细胞 - 细胞外基质粘附的研究是一个研究热点,也是许多生物学领域广泛关注的课题。

除了其他多种技术之外,我们(Georgiadou等人,2017; Lilja等人,2017;Närvä等人,,2017)和其他人(Kiely等人,2015;Böhm等人,2016; Salmela等人,2016)已经使用xCELLigence RTCA系统作为监测细胞ECM粘附的变化的简单而定量的方法。该技术通过测量在导电溶液如组织培养基(图1)存在下融合到微量滴定板底部表面的金微电极之间传输的电子流来工作。粘附的细胞破坏了电极与本体溶液之间的相互作用,从而阻碍电子流动。这种阻抗(抗交流电)表示为称为细胞指数的任意单位(图1),其大小取决于细胞数量,细胞形态和细胞大小,以及细胞附着于板上基质的强度。与传统的基于染料或显微镜的细胞粘附分析相反,使用xCELLigence RTCA的优势人,2009年;汉弗莱斯,2009; Humphries等人>,2009;陈,2012)是从细胞开始附着到基质的时刻开始,可以获得细胞粘附的连续读数,而不是单个时间点或终点分析。此外,测量是基于整个细胞群而不是单独选择的细胞,因此结果不太可能受到偏差。但是,有分析基于细胞群的粘附性有缺陷的情况。例如,当研究蛋白质过表达或敲低的影响并且转染效率非常低时,xCELLigence系统将不能真实反映对细胞粘附的实验操作。事实上,在这些情况下,在个体转染的细胞中,基于显微镜的已知粘附成分的成像,以监测细胞-ECM接触的大小和/或形态学的变化,以及肌动蛋白细胞骨架,以监测细胞大小和细胞扩散,将是更多合适的方法。尽管如此,在xCELLigence RTCA单板(SP)模型中包含96孔E形板格式,可以同时在同一个平板上同时测试多个实验条件,从而降低了实验的可变性,并可以快速推导出最佳分析方法参数(例,ECM配体浓度或时间点/粘附时间)进行更全面的分析,这可能需要更昂贵的试剂和额外的优化。

关键字:xCELLigence, 实时细胞分析, 细胞粘附, 细胞外基质, 整合素

材料和试剂

  1. 移液器提示
  2. 组织培养物处理的培养皿(CELLSTAR 100×20mm,Greiner Bio One International,目录号:664160; 6孔,Greiner Bio One International,目录号:657160; 96孔,Greiner Bio One国际,目录号:655160)
  3. Falcon 15-ml锥形离心管(Corning,Falcon ,目录号:352196)
  4. Falcon 50-ml圆锥形离心管(Corning,Falcon ,目录号:352070)
  5. 微量离心管,1.5毫升(SARSTEDT,目录号:72.690.001)
  6. Minisart 0.45μm一次性过滤器(Sartorius,产品目录号:16537-K)
  7. 60毫升注射器(BD,目录号:300866)
  8. 96孔E板(E-plate 96)(ACEA Bio,目录号:5232368001)
  9. 感兴趣的细胞系,例如HEK293细胞(ATCC,目录号:CRL-1573)
  10. 磷酸盐缓冲液(PBS)(Sigma-Aldrich,目录号:D1408)
  11. 例如ECM分子,纤连蛋白(牛血浆)(Sigma-Aldrich,目录号:341631);胶原蛋白(来自小牛皮肤的胶原)(Sigma-Aldrich,目录号:C8919)
  12. HyClone TM HyQTase细胞分离试剂(GE Healthcare,HyClone TM,目录号:SV30030.01)
  13. Dulbecco改良的含高葡萄糖(4500mg / L)的Eagle培养基(DMEM)(Sigma-Aldrich,目录号:D5671)
  14. L-谷氨酰胺(Thermo Fisher Scientific,Gibco TM,目录号:25030149)
  15. 胎牛血清(FBS)(Sigma-Aldrich,目录号:F7524)
  16. 用于细胞培养的胰蛋白酶-EDTA(Sigma-Aldrich,目录号:T4049)
  17. ≥96%纯牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A8022)
  18. 用于培养感兴趣的细胞系的适当生长培养基(见食谱)

  19. 在实验中使用适当的基础培养基(见食谱)
  20. 纤连蛋白 - 胶原蛋白混合物(见食谱)
  21. 0.1%BSA(见食谱)

设备

  1. 可调容量移液器(例如,Fisher Scientific,型号:Fisherbrand TM Elite TM)
  2. 水浴
  3. 多通道移液器(例如,Fisher Scientific,Fisher ScientificTM,FisherbrandTM TM Elite TM)
  4. 37℃,5%CO 2水夹套培养箱(例如,Thermo Fisher Scientific,Thermo Scientific TM,型号:8000系列水夹套CO 2培养箱)
  5. 细胞培养层罩(例如,NuAire CellGard TM)
  6. 用于15ml和50ml锥形管(例如Eppendorf,型号:5804)的台式离心机。
  7. Bürker细胞计数室(,例如,BRAND,目录号:719520)
  8. 明场显微镜(例如,奥林巴斯,型号:CKX41或ZEISS Axio Vert)
  9. xCELLigence RTCA SP仪器(ACEA Bio,完整系统的产品目录号:00380601030)包括:
    1. RTCA分析仪(ACEA Bio,型号W830,产品目录号:05228972001) - 一种电子分析仪,用于测量,处理和分析传感器电极检测到的阻抗。
    2. RTCA SP Station(ACEA Bio,产品目录号:05229057001) - 置于培养箱内的E板支架,将E板连接至RTCA分析仪。
    3. RTCA控制单元(ACEA Bio,目录号:05454417001) - 带预装RTCA软件的笔记本电脑

软件

  1. RTCA软件(版本号1.2.1.1002)
  2. Microsoft Excel
  3. GraphPad Prism 6(版本6.05)

程序

注意:

  1. 在无菌条件下执行所有步骤。
  2. 避免触摸检测器所在平板的下侧。
  3. 避免划伤位于井底的电极。这可以通过移液管吸头与电极接触而发生。去除溶液时,稍微倾斜平板,将吸管放在孔的一侧,轻轻吸取溶液。















  4. 使用至少3-4个井作为技术上的重复测试每个条件
  5. 在计数/接种之前,通过充分混合细胞悬液并确保足够的细胞 - 细胞解离,避免E-plate 96孔中细胞计数和/或细胞结块的错误。在适当的情况下使用多道移液器,以确保每个孔都添加细胞。
  6. 优化每个待测细胞系的细胞数量;为了监测细胞与ECM的粘附,最好避免细胞/汇合单层过度拥挤。
  7. E-plate 96每孔的最大推荐体积为200μl。
  8. 有关更多信息,请参阅故障排除指南(请参阅Notes部分中的表1)。

  1. 准备电子盘
    1. 用150μl无菌PBS冲洗孔,然后吸出缓冲液。
    2. 用100μlECM分子或100μl0.1%BSA(阴性对照,参见食谱)在37℃下包被1小时。
      注:ECM涂层的选择取决于所使用的电池。对于细胞粘附力弱的HEK293细胞,我们过去使用纤连蛋白和胶原ECM分子的混合物(参见食谱)。
    3. 去除涂层,用150μlPBS洗两次。
    4. 通过将所有孔与100μl0.1%BSA在37℃下孵育1小时,阻断非特异性细胞与E板的结合。
    5. 去除BSA,用150μlPBS洗两次。
    6. 在每个孔中加入50μl预热的基础培养基(参见配方),并在开始实验之前将E-plate留在培养箱(37℃)中至少15分钟,以确保培养基和E-板面达到平衡。

  2. 设置xCELLigence RTCA程序
    注意:程序设置需要在开始实验之前完成。
    1. 启动RTCA程序。
    2. 设置实验注释页面(图2A) - 选择要保存实验文件的文件目录,填入实验信息,如实验日期,细胞系和处理,实验程序,一个目的和任何额外的信息,你想保存。
    3. 设置 Layout 页面(图2B)。该页面记录运行的实验布局。请注意,未标记的井将不会被扫描。一次选择一个或多个孔(复制)(选定的孔将被高亮显示)。输入每个孔的适当信息,例如细胞类型,细胞数量,使用的化合物等。 例如:Cell Type-HEK293,control,Cell number-20,000。


      图2.设置xCELLigence RTCA程序 - 实验细节 “注”(A)和 Layout 显示了用HEK293细胞进行的实验。每个条件包括四个技术重复。选定的蓝色井的详细信息可以在页面顶部看到。

    4. 为平板运行程序设置 Schedule 页面(图3)。实验可以分为多个步骤,其中包含一个或多个扫描。一次扫描包括对所有选择的孔进行一次扫描(每个孔一次)。
      1. 步骤1被认为是背景(基线)测量,即在添加细胞(孔仅含有基础培养基)之前对E板进行扫描。第一步预编程为一次扫描。不要更改第1步的设置。
      2. 每次使用添加步骤选项(图3)从RTCA仪器中移除E盘时,请单独制作一个 Step >步骤2 添加细胞,步骤3 添加化合物)。
      3. 如果要以不同的速率测量事件,请使用添加子步骤并编辑间隔来连续扫描, / em>和扫描,方法是输入适当的值并单击应用。在下面的例程中,程序会在 Step 1 后暂停,但是会自动从步骤2.1 (子步骤)移动到步骤2.2 <子步骤需要手动确认。扫描之间的间隔越短,对于检测快速和潜在的短期事件,例如初始细胞附着到基质,都是有用的。
        为HEK293细胞设立的一个例子:
        1. 第1步(背景测量); 1扫(总时间00:00:06)。
        2. (,例如,子步骤监视初始单元格连接到底层)。 16次扫描,间隔2分钟(总时间为00:30:06)。
        3. (例如,子步骤用于监测随后的细胞铺展和对基质的粘附)。 18次扫描,间隔5分钟(总时间02:00:06)。
        为MEF设立的一个例子:
        1. 第1步(背景测量); 1扫(总时间00:00:06)。
        2. 第二步(监视细胞附着和扩散); 18次扫描,间隔10分钟(总时间03:00:06)。
        注意:优化感兴趣细胞系的扫描次数和间隔时间非常重要。为了避免浪费E板,首先使用正常的96孔板来发现细胞粘附的时间线,即当一些细胞粘附非常快(几分钟)并在2小时内扩散时,其他细胞可能需要更长的时间来粘附传播(3-4小时)。当首次进行xCELLigence粘附实验时,可以使用较短的时间间隔(如上述步骤2.1)1小时监测细胞指数,然后相应调整下一个实验的参数。

        图3.设置xCELLigence RTCA程序时间表 - 步骤和扫描。 HEK293单元显示了 Schedule 页面的示例。步骤1(A)是预编程的,不应该改变。使用添加步骤和/或添加子步骤(A)在计划(B)中插入具有不同扫描和间隔的其他实验步骤。

  3. 使用xCELLigence RTCA背景阅读执行细胞粘附实验
    1. 一旦程序设置完毕(见上文),将预先准备好的预热的含有50μl基础培养基的E-plate放入RTCA SP站。
    2. 点击开始开始实验。这将启动 Step 1 来测量基本介质的背景阻抗。这个读数被用作细胞指数值的参考阻抗。
    3. 现在, Step-Status 会显示 TEST 。检查消息页面,了解被测井的状态和连接是否正常。
      重要提示:排除任何会导致读数异常的井。基线读数预计接近于零。请参阅故障排除部分(表1)。
    4. 完成 Step 1 后, Step-Status 显示 DONE ,程序已准备好进行下一步。
      注意:RTCA程序不会进入下一步,直到手动指示为止。
    5. 从RTCA SP Station上取下E盘,然后继续添加单元格。

  4. 使用xCELLigence RTCA加入细胞进行细胞粘附实验
    1. 去除HEK293细胞的生长培养基(见食谱)。用5毫升的无菌PBS洗细胞。
    2. 去除PBS,加入2毫升预热的HyQtase(体积调整为汇合的10厘米培养皿),并在37°C孵育板,直到所有的细胞已经分离。
    3. 加入5ml的基础培养基,然后通过离心(180×g,4分钟)收集细胞。
      注意:如果使用胰蛋白酶分离细胞,建议使用胰蛋白酶抑制剂或加入含有血清的5ml生长培养基来抑制胰蛋白酶活性。我们推荐使用HyQtase或其他类似的产品,而不是像胰蛋白酶这样的消化酶来分离整合素功能正在研究的短期测定的细胞。整合素对胰蛋白酶切割敏感,因此使用胰蛋白酶可影响细胞粘附到涂覆的E板上所需的时间。在一些情况下,当细胞非常粘附时,使用胰蛋白酶可能是不可避免的,并且ECM降解需要破坏细胞-ECM连接。
    4. 弃去上清液并将剩余的细胞沉淀重新悬浮在4ml的基础培养基中。
      重要提示:多次上下轻轻打结,打破细胞团块。
      注意:如果使用生长培养基抑制胰蛋白酶,离心后确保完全除去培养基,用无血清基础培养基重新洗涤沉淀,重复上述离心步骤。
    5. 使用所选择的方法(例如,Bürker细胞计数室)对细胞进行计数,制备2×10 5个细胞/ ml(在基础培养基中稀释)的细胞悬液并转移100μl(20,000个细胞;针对HEK293细胞系进行了优化)到预先制备的E-plate的每个孔(已经含有50μl的基础培养基;因此每个孔的总体积现在为150μl)。
      注意:细胞数量必须针对每个感兴趣的细胞系进行优化,以便细胞有足够的空间来粘附和扩散。建议使用正常组织培养处理的96孔板和明场显微镜进行这种优化,因为E-plate 96更昂贵。我们过去在xCELLigence细胞粘附测定中使用了以下细胞和细胞数目:MEF和人端粒酶永生化成纤维细胞:7,500-10,000; MDA-MB-231细胞:15,000-20,000;人类iPSC:50,000。但是,这些只是指导原则。不同的实验处理或培养条件可能影响细胞存活或细胞大小,因此最好相应地优化细胞数量。
      重要提示:执行大量条件时,单元格添加到井的顺序可能会影响数据。例如,如果对照细胞粘附非常快,并且您预期处理的细胞将响应更慢,则如果始终将对照细胞加入孔中并且经过几分钟,然后可以对处理的细胞进行铺板,则这种差异可能被夸大。为了克服这个潜在的问题,尽可能地尝试使用多道移液器以加速细胞添加,并尝试改变细胞添加到生物重复中的孔的顺序,以查看数据是否受到细胞添加顺序的影响。如果这似乎是这种情况,考虑减少实验的复杂性,以减少细胞之间的电镀间隔。
    6. 将E-plate重新插入RTCA SP Station,然后点击 Start 以扫描 Step 2 。
      重要提示:分析数据时,步骤2的第一次测量应被视为时间点0。
    7. 在实验过程中,您可以按照 Plot 页面实时查看细胞索引。为此,请选择感兴趣的孔/孔,然后单击添加。通过单击添加所有实验中包含的每个孔都被绘制出来。
    8. 完成实验后,打开 Plot 页面,点击全部添加,然后选择一个全时标度,如果您希望收集整个实验的数据。如果点击平均值,复制孔(名称相同的孔)将被合并,只导出平均单元格索引值。通过选择 Plate-Export Experiment Info ,将原始数据(单元格索引与时间)导出到Excel电子表格中。

数据分析

  1. 对于实验设计,为了比较两个数据集,我们进行至少三次独立时间的xCELLigence粘附分析。对于每个实验,我们至少使用三个技术重复。
  2. 细胞粘附作为实时平均细胞指数值的变化呈现(见图4的图解说明)。我们通常观察到技术重复的范围为0.001-0.15的标准偏差(SD),以及n = 4生物重复的0.02-0.2的标准偏差(最高的SD出现在较晚的时间点)。我们使用GraphPad Prism和Student's t -test在两个数据集的终点单元格索引值之间执行统计分析。


    图4.来自两个数据集的xCELLigence细胞粘附结果的分析 xCELLigence RTCA数据的实例分析显示细胞粘附作为平均细胞指数±SEM与时间的关系。这里,时间点0表示电池添加之后E板96首先被扫描的点。 E-plate 96的初始扫描( Step 1 )用于在没有细胞的情况下监测背景读数,不包含在分析中。对两个数据集之间的终点单元索引值进行统计分析(用浅紫色框突出显示)。

笔记

  1. 细胞粘附测定相对简单且可重现。然而,与该测定相关的一个常见问题是,细胞可能在播种后积累在井的中间而不是整个井中。这将导致不准确的细胞指数值和实验之间的变化增加。在xCELLigence手册中,建议在细胞接种后将平板保持在室温30分钟以使细胞沉降到平板的底部;然而,这不是细胞粘附测定的选择,因为细胞会立即开始粘附,因此这些早期粘附事件的记录将会丢失。因此,电镀完成后尽快将印版送回RTCA SP工作台E板固定器是非常重要的,并立即开始 Step 2 。正如我们的协议中所述,我们建议预加热E板,在电镀之前和电镀之后,如果可能的话使用多通道移液器进行中等和彻底的细胞混合。此外,一旦细胞已经播种,我们已经发现,在顺时针方向顺时针方向上平板上的轻微搅动有助于使细胞更均匀地分散在整个孔中。尽管如此,如果第一次进行测定,或者如果使用新细胞系,特别是上皮细胞或内皮细胞,我们推荐使用正常组织培养处理的96孔板来优化细胞数字和细胞添加技术。
  2. 这里使用的电子版不适合亮视野成像。尽管可以购买专门的电子版用于获得相位差图像,但是这些成本更高。此外,除了预先设定的步骤(例如药物添加)之外,不建议停止RTCA程序或长时间移除E板。如果需要对细胞进行目视检查,我们推荐使用正常组织培养处理的96孔板,以完全相同的方式(即,相同的涂层,阻塞和细胞数量)设置,并与xCELLigence粘附分析平行进行。
  3. 疑难解答指南(表1)

    表1. xCELLigence细胞粘附分析的故障排除指南。 该表格包含用户在分析过程中可能遇到的一些常见问题的建议。但是,这张表并不是详尽的,如果需要的话,用户应该参考制造商的手册以获得进一步的建议。

食谱

  1. HEK293生长培养基
    445毫升无菌达尔伯克氏改良伊格尔培养基(DMEM)与高葡萄糖
    5毫升无菌200毫米L-谷氨酰胺
    50毫升无菌胎牛血清(FBS)
    在4°C储存长达1个月
  2. HEK293基础培养基(无血清生长培养基)
    495毫升无菌DMEM
    5毫升无菌200毫米L-谷氨酰胺
    在4°C储存长达1个月
  3. ECM分子的选择
    纤连蛋白 - 胶原蛋白混合物:

    用无菌PBS稀释无菌原液制备10μg/ ml纤连蛋白和20μg/ ml胶原的混合物 重要:不要过滤工作溶液,因为这会导致过滤器上ECM分子的损失。只使用新制备的工作解决方案。
    注意:对于具有不同粘附特性的其他细胞系,如果使用其他ECM蛋白,建议首先测试您的特定实验的最佳ECM配体浓度(图1)。
  4. 0.1%BSA
    在100ml无菌PBS中稀释0.1g的96%BSA并过滤灭菌 只使用新鲜准备的工作解决方案

致谢

这项工作得到了芬兰科学院,ERC合并补助金(615258),Sigrid Juselius基金会和芬兰癌症组织的资助。 J.L.由图尔库分子医学博士课程(TuDMM)提供支持。该协议是根据Georgiadou等人(2017),Lilja等人(2017),Närvä等人发表的程序改编的。 (2017)。作者宣称没有利益冲突。

参考

  1. Böhm,M.,Apel,M.,Lowin,T.,Lorenz,J.,Jenei-Lanzl,Z.,Capellino,S.,Dosoki,H.,Luger,TA,Straub,RH和Grässel,S. 2016)。 α-MSH调节骨关节炎患者的滑膜成纤维细胞的细胞粘附和炎症应答 Biochem Pharmacol 116:89-99。
  2. Bökel,C.和Brown,N.H。(2002)。 整合研发:继续,响应和坚持细胞外基质。 Dev Cell 3(3):311-321。
  3. Bouvard,D.,Pouwels,J.,De Franceschi,N.和Ivaska,J。(2013)。 整合素失活剂:在体内平衡细胞功能 和 。 Nat Rev Mol Cell Biol 14(7):430-442。
  4. Calderwood,D.A.,Campbell,I.D。和Critchley,D.R。(2013)。 Talins和kindlins:整合素介导的粘附的合作伙伴 Nat Rev Mol细胞生物学 14(8):503-517。
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  7. Georgiadou,M.,Lilja,J.,Jacquemet,G.,Guzman,C.,Rafaeva,M.,Alibert,C.,Yan,Y.,Sahgal,P.,Lerche,M.,Manneville,JB,Makela TP和Ivaska,J。(2017)。 AMPK负向调节张力蛋白依赖性整联蛋白活性 J细胞生物学
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  9. Horton,ER,Byron,A.,Askari,JA,Ng,DHJ,Millon-Fremillon,A.,Robertson,J.,Koper,EJ,Paul,NR,Warwood,S.,Knight,D.,Humphries,JD和Humphries,MJ(2015)。 粘连复合体装配和拆卸过程中共有整合素粘附体的定义及其动力学 Nat Cell Biol 17(12):1577-1587。
  10. Humphries,M. J.(2009)。 细胞粘附分析。 Methods Mol Biol 522:203 -210。
  11. Huveneers,S.and Danen,E.H。(2009)。 粘附信号 - 整联蛋白,Src和Rho之间的串扰
  12. Humphries,J. D.,Byron,A.,Bass,M.D.,Craig,S.E.,Pinney,J.W.,Knight,D.and Humphries,M.J。(2009)。 整联蛋白相关复合物的蛋白质组学分析将RCC2鉴定为Rac1和Arf6的双重调节剂。 Sci Signal 2(87):ra51。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Hamidi, H., Lilja, J. and Ivaska, J. (2017). Using xCELLigence RTCA Instrument to Measure Cell Adhesion. Bio-protocol 7(24): e2646. DOI: 10.21769/BioProtoc.2646.
  2. Georgiadou, M., Lilja, J., Jacquemet, G., Guzman, C., Rafaeva, M., Alibert, C., Yan, Y., Sahgal, P., Lerche, M., Manneville, J. B., Makela, T. P. and Ivaska, J. (2017). AMPK negatively regulates tensin-dependent integrin activity. J Cell Biol 216(4): 1107-1121.
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