Visualizing Loss of Plasma Membrane Lipid Asymmetry Using Annexin V Staining

Materials and reagents

1. Mammalian cell culture

   In this study, we used mouse myoblast cells (C2C12; cell number: ACC 565, DSMZ Braunschweig, Germany) and the corresponding knockout cells lacking CDC50A (Grifell-Junyent et al., 2022) that were cultured in growth medium (see Recipe 1). Optimal culture media and conditions may differ for other cell lines.

   1. Basal cell culture medium for growth (e.g., high glucose DMEM, without pyruvate; Sigma-Aldrich, catalog number: D5796), store at 4 °C

   2. Ethanol absolute ≥ 99.8% (VWR, catalog number: 20821.321)

   3. Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) (e.g., Sigma-Aldrich, catalog number: E4378)

   4. Fetal bovine serum (FBS), heat inactivated before use (e.g., Capricorn Scientific, catalog number: FBS-11A), store at -20 °C

   5. Hanks’ balanced salt solution, Ca²⁺ and Mg²⁺ free (HBSS) (e.g., Sigma-Aldrich, catalog number: H6648), store at 4 °C

   6. 35 mm polymer bottom dishes (e.g., Ibidi, catalog number: 81156)

   7. 1.5 mL microcentrifuge tubes (Sarstedt, catalog number: 72.690.001)

   8. Penicillin-streptomycin, 100× solution (e.g., Sigma-Aldrich, catalog number: P4333), store at -20 °C

   9. Pipette controller (e.g., accu-jet pro, Brand, catalog number: 263 00)

   10. Polypropylene tubes, 15 mL capacity (e.g., Falcon tubes, Sarstedt, catalog numbers: 62.554.502 and 62.547.254)

   11. Sterile serological pipettes (e.g., Serological pipettes of 5, 10, and 25 mL; Sarstedt, catalog numbers: 86.1253.001, 86.1254.001, and 86.1685.001)

   12. Sterile culture vessels T-75 flasks (e.g., Sarstedt, catalog number: 83.3911)

   13. Trypsin-EDTA solution (e.g., Sigma-Aldrich, catalog number: T3924), store at -20 °C

   14. Trypan Blue solution, 0.4% (e.g., Thermo Fischer Scientific, catalog number: 15250061)

   15. Growth medium (see Recipe 1)

       
       

2. For annexin V labeling

   1. Annexin V conjugated to Alexa Fluor 568 (e.g., Roche, catalog number: A13202), store at 4 °C

   2. 4’,6-Diamidino-2-phenyl-indol-dihydrochlorid (DAPI) (e.g., Sigma-Aldrich, catalog number: D9542)

   3. Dead cell staining reagents, e.g., SYTOX Blue (Thermo Scientific, catalog number: S34857)

   4. Ice

   5. Tyrode’s balanced salt solution with Ca²⁺ (TBSS + Ca²⁺; see Recipe 2), store at 4 °C

   6. Tyrode’s balanced salt solution without Ca²⁺ (TBSS - Ca²⁺; see Recipe 3), store at 4 °C

   7. DAPI stock solution (1 mg/mL) (see Recipe 4)

      Note: This procedure has also been successfully performed using FITC-labeled lactadherin (e.g., Haematologic Technologies, catalog number: BLAC-FITC).

      
      

3. For the preparation of giant unilamellar vesicles (GUVs)

   1. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (Avanti^® Polar Lipids, catalog number: 850375)

   2. 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (Avanti^® Polar Lipids, catalog number: 850725)

   3. 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DOPS) (Avanti^® Polar Lipids, catalog number: 840035)

   4. Calcium chloride (CaCl₂) (Grüssing, catalog number: 10043-52-4)

   5. Chloroform, ethanol-stabilized and certified for absence of HCl (Sigma-Aldrich, catalog number: 32211-M)

   6. Detergent/soap

   7. Ethanol, 70% (Sigma-Aldrich, catalog number: 64-17-5)

   8. Glucose (Duchefa Biochemie, catalog number: G0802.5000)

   9. HEPES (Carl Roth, catalog number: 7365-45-9)

   10. Ice

   11. Magnesium chloride hexahydrate (MgCl₂·6H₂O) (Sigma-Aldrich, catalog number: 7791-18-6)

   12. Methanol ≥ 99.8% (VWR, catalog number: 67-56-1)

   13. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 1310-58-3)

   14. Potassium chloride (KCl) (Merck, catalog number: 7447-40-7)

   15. Sodium chloride (NaCl) (Carl Roth, catalog number: 7647-14-5)

   16. Sucrose (Duchefa Biochemie, catalog number: S0809.5000)

   17. Lipid stock in chloroform (see Recipe 5)

   18. Swelling buffer (320 mM sucrose) (see Recipe 6)

   19. Binding buffer (see Recipe 7)

   Note: Store or keep all reagents at room temperature, except indicated items. All buffers are prepared the day before and stored at 4 °C.

Equipment

1. General

   1. Analytical balance (e.g., Sartorius Entris-I II, 220 g/0.1 mg; Buch Holm, catalog number: 4669128)

   2. Computer with monitor (e.g., DELL U2415)

   3. Confocal laser scanning microscope (e.g., Leica TCS SP8 confocal laser scanning microscope, Leitz, Wetzlar, Germany, equipped with a 63×/1.20 water objective)

   4. Eppendorf Research^® plus pipettes P20, P200, P1000 (Eppendorf, catalog numbers: 3123000039, 3123000055, 3123000063)

   5. Eppendorf tube, 2 mL (Merck, catalog number: EP0030120094)

   6. Freezers -20 °C and -80 °C

   7. Magnetic stirrer (e.g., IKAMAG^®, DREHZAHL ELECTRONIC, IKA, Staufen im Breisgau, Germany)

   8. Magnets

   9. pH meter (pH-Meter 761 Calimatic, Knick, Berlin, Germany)

   10. Pipette tips 10, 200, 1,000 μL (Sarstedt, catalog numbers: 70.760.002, 70.3030.020, 70.3050.020)

   11. Refrigerator (5 °C)

   12. Water distillation system

       
       

2. For cell culture

   1. Autoclave sterilizer (e.g., Systec VX-65, Systec, Linden, Germany)

   2. Biological safety cabinet certified for handling biological materials (e.g., Herasafe KSP Class II Biological Safety Cabinets, Thermo Fisher Scientific)

   3. Centrifuge with rotor for 15 and 50 mL polypropylene tubes (e.g., Eppendorf 5810 R; Wesseling, Germany)

   4. Incubator with humidity and gas control to maintain 37 °C and 95% humidity in an atmosphere of 5% CO₂ in air (e.g., Binder, Tuttlingen, Germany)

   5. Inverted phase contrast microscope equipped with a 10× objective (HI PLAN I 10×/0.22 PH1; Leica DMi1, Mannheim, Germany)

   6. Neubauer counting chamber (improved dark lines, 0.1 mm) and cover glasses (20 mm × 26 mm × 0.4 mm)

   7. Water bath (e.g., WPE45 Memmert, Schwabach, Germany) for mammalian cells and for NBD-lipid labeling (Julabo CORIO C-BT5, catalog number: 9011305)

      
      

3. For preparation of GUVs

   1. Cover glass slides (26 mm × 76 mm, #1.5, Thermo Fisher Scientific, Life Technologies Corporation Eugene)

   2. Flow cabinet to work with organic solvents

   3. Glass beads, 3 mm (Merck, catalog number: 104015)

   4. Glass desiccator Boro 3.3 with a socket in the lid, 20 cm, including stopcock (Brand, catalog number: 65238)

   5. Glass pipettes (e.g., graduated pipettes BLAUBRAND^® Type 3 Class AS, 10 mL, graduation: 10 mL; Carl Roth, catalog number: HXT8.1)

   6. Glass slide (Thermo Scientific, microscope slides 76 mm × 26 mm, catalog number: MEZ 101026)

   7. Glass vials (Rotilabo^® screw neck ND8 vials, brown/white glass, 1.5 mL; Carl Roth, catalog number: KE30.1) with screw caps (without a borehole, without septum, PP, black, ND8; Carl Roth, catalog number: KE39.1)

   8. Glass tubes (Carl Roth, catalog number: DURAN C208.1)

   9. Hamilton 700 Series syringes 25, 100, 1,000 μL (Hamilton Company, Nevada, USA)

   10. High vacuum grease (DOW CORNING, 65201 Wiesbaden, made in USA, Artwork Nr. 0315)

   11. Ice bucket (e.g., Magic Touch 2^TM ice bucket with lid; Sigma-Aldrich, catalog number: BAM168072002)

   12. O-ring (28 mm × 1 mm, Nanion Technologies, München)

   13. Parafilm (PARAFILM^® M; Sigma-Aldrich, catalog number: P7793-1EA)

   14. Rotavapor^® R-100 Evaporator with I-100 Controller and V-100 vacuum pump (Flawil, Switzerland)

   15. Scissors

   16. Ultra-violet/ozone probe and surface decontamination unit (e.g., Novascan Technologies Inc., Boone, IA, USA)

   17. Vortex mixer (e.g., Vortex Genie 2 Scientific Industries Inc., catalog number: SI-0236)

   18. Vacuum Pump V-100 with Interface I-100 (Buchi, catalog numbers: 11593636 and 11593655D)

   19. Wipes (Precision Wipes, KIMTECH Science, Kimberly-Clark^® Professional, catalog number: 7552)

Software

1. ImageJ (Wayne, Rasband, S., U. S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/index.html , version v.153q)

2. Leica Application Suite AF (LAS AF, Leitz, Wetzlar, Germany)

3. Microsoft Excel (Microsoft Corporation, 2018)

4. PowerPoint (Microsoft Corporation, 2018)

5. OriginPro (OriginLab, 2023)

Procedure

The following procedure outlines four main steps: (A) preparation of mammalian cells, (B) cell counting, (C) annexin V staining of adherent cells, and (D) preparation of GUVs. The last step was originally applied to study the binding specificity of annexin V to different membrane lipid compositions (Weingärtner et al., 2012; Grifell-Junyent et al., 2022). We recommend this analysis before applying new probes in cell studies.

1. Preparation of mammalian cells

   1. Grow adherent cells in sterile culture vessels (T-75 flask) in growth medium (see Recipe 1) in a tissue culture incubator (37 °C, 5% CO₂, 95% humidity) until they reach ~60%–70% confluency.

      Caution: C2C12 cells will differentiate if grown too confluent and start to fuse. Differentiation and fusion are accompanied by transcriptional changes, which can lead to different results.

   2. Aspirate and discard growth media with sterile serological pipette.

   3. Wash cells twice with 5 mL of HBSS (Ca²⁺ and Mg²⁺ free, pre-warmed at 37 °C) using a sterile serological pipette.

   4. Add 1.5 mL of trypsin-EDTA solution (pre-warmed at 37 °C) using a sterile serological pipette and incubate the T-75 flasks in a tissue culture incubator (37 °C, 5% CO₂, 95% humidity). Tilt the vessel back and forth a few times to make sure the thin layer of trypsin is evenly spread.

   5. After 5 min, check for detachment by gently tilting vessel and/or observing under the microscope. If all cells have not detached in 5 min, incubate an additional 1–2 min and check again. Continue to incubate and check as necessary, only until cells are no longer attached to the plate surface.

      Caution: Avoid prolonged incubation period with trypsin-EDTA solution.

   6. Stop trypsinization by adding 7.5 mL of growth medium (pre-warmed at 37 °C, see Recipe 1) to the cell suspension.

   7. Transfer the cell suspension into a 15 mL Falcon tube and set aside 100 μL in a 1.5 mL microcentrifuge tube for cell counting, e.g., using the hemocytometer (see section B).

   8. Centrifuge cells in the 15 mL Falcon tube at 300× g for 5 min at room temperature and discard the supernatant to remove the trypsin-EDTA containing medium from the cells.

   9. Add 10 mL of fresh growth medium (pre-warmed at 37 °C, see Recipe 1) to the cell pellet and re-suspend completely by gently pipetting up and down using a serological pipette.

      Caution: Cells in suspension settle quickly. After counting, we recommend gently re-suspending the cell suspension approximately every 2–3 min when seeding multiple dishes.

   10. After counting (see section B), seed 1.5 × 10⁴ cells per 35 mm polymer bottom dishes and add growth medium (pre-warmed at 37 °C, see Recipe 1) up to a final volume of 1 mL per dish.

       Note: Prepare sufficient Petri dishes for control samples (see section C). We used a low cell number for seeding because C2C12 is a fast-growing cell line (doubling time: ~20 h) and this cell number guarantees that single cells are still present when the assay is performed the next day.

   11. Keep the cells in a tissue culture incubator (37 °C, 5% CO₂, 95% humidity) overnight.

       
       

2. Cell counting (supplemental; if information on cell counting is not required, proceed to section C)

   The purpose of this step is to quantify the cell concentration to resuspend the cells at the appropriate concentration for the assay. We routinely use the trypan blue hemocytometer assay. Alternative cell counting methods such as automatic cell counters may be used.

   1. Prepare the hemocytometer by cleaning the chambers and coverslip with ethanol. Dry the hemocytometer by using lint-free tissue. Place the glass coverslip over the counting chambers.

      Note: The correct placement is indicated by the appearance of the Newton rings.

   2. Add 100 μL of 0.4% trypan blue solution to 100 μL of cell suspension (step A7) to obtain a 1:1 dilution using a P200 Eppendorf pipette.

   3. Load the hemocytometer with 10 μL of cell suspension per counting chamber with a P20 Eppendorf pipette and examine immediately under an inverted phase contrast microscope at low magnification (e.g., 5–10× magnification).

      Critical: To ensure accurate results, it is important to avoid over- or underfilling the cell suspension chamber.

   4. Count the number of viable (seen as bright cells) and non-viable cells (stained blue) in the large outer quadrants.

   5. Calculate the percentage of viable cells: % viable cells = [1.00 - (number of blue cells ÷ number of total cells)] × 100. Cell viability should be at least 95%.

   6. Calculate the cell concentration based on the premise that each square accounts for a volume of 10^-4 mL of cell suspension.

   7. To obtain the total number of viable cells per milliliter of aliquot, multiply the total number of viable cells by 2 (the dilution factor for trypan blue) and the correction factor of 10⁴ (volume of each square).

      
      

3. Annexin V staining of adherent cells

   We suggest that at least four samples are prepared (Figure 1): i) a negative control without staining to determine background fluorescence, ii) a sample stained with DAPI only, iii) a sample to be stained with DAPI and annexin V in the presence of calcium, and iv) a sample to be stained with DAPI and annexin V in the absence of calcium.

   
   

   
   Figure 1. Schematic illustration of the annexin V binding assay. Preparation of a negative control without DAPI and annexin V staining, a control with DAPI-stained cells, and cells stained with DAPI and annexin V conjugated to Alexa Fluor 568 for 10 min on ice with TBSS with or without calcium, respectively. The negative control without staining is used to assess the level of background fluorescence in the sample. This is important because even in the absence of a fluorescent stain, there may still be some level of background fluorescence present due to autofluorescence or other sources. DAPI staining (at a final concentration of 10 μg/mL) is used for visualization of nuclei and cell counting. Non-specific binding of Ca²⁺-dependent annexin V is tested in the same assay by using TBSS without Ca²⁺.

   
   

   1. Prior to the start of the assay, prepare TBSS with and without calcium (± Ca²⁺; see Recipes 2 and 3) and a DAPI stock solution (see Recipe 4).

      Note: Annexin V requires the presence of Ca²⁺ to bind to PS. TBSS without Ca²⁺ is used as a negative control.

   2. To prepare the cells for labeling, carefully aspirate and discard the growth medium using a P1000 Eppendorf pipette.

   3. Wash cells twice with 1 mL of TBSS (± Ca²⁺; cooled on ice, see Recipes 2 and 3) using a P1000 Eppendorf pipette.

   4. After washing, add 0.5 mL of TBSS (± Ca²⁺; cooled on ice, see Recipes 2 and 3) using a P1000 Eppendorf pipette.

   5. Add 5 μL of the annexin V conjugated to Alexa Fluor 568 using a P20 Eppendorf pipette and incubate for 10 min on ice in the dark.

      Note: Skip steps C5–C8 for the negative control (i); skip steps C5–C7 for the sample only stained with DAPI (ii).

   6. Remove staining solution and wash the cells twice with 1 mL of TBSS (± Ca²⁺; cooled on ice, see Recipes 2 and 3) using a P1000 Eppendorf pipette.

   7. After washing, add 0.5 mL of TBSS (± Ca²⁺; cooled on ice, see Recipes 2 and 3) using a P1000 Eppendorf pipette.

   8. Add DAPI stock solution to a final concentration of 10 μg/mL (stock 1 mg/mL: add 5 μL using a P20 Eppendorf pipette). Incubate the cells at room temperature for 10 min in the dark.

      Note: At the concentration used here, DAPI stains the nucleus of both live and dead cells and is used for the visualization of nuclei and cell counting. When using DAPI for dead cell staining, a final concentration of 0.1 μg/mL is recommended. Alternatively, other dead cell staining reagents such as SYTOX Blue can also be used.

   9. Remove staining solution and wash cells twice with 1 mL of TBSS (± Ca²⁺; cooled on ice, see Recipes 2 and 3) using a P1000 Eppendorf pipette.

   10. Add 1 mL of TBSS (± Ca²⁺; cooled on ice, see Recipes 2 and 3) using a P1000 Eppendorf pipette and image on the microscope.

       
       

4. Preparation of GUVs

   1. Prepare the stock and working solutions of the lipids

      1. Clean the Hamilton syringes by flushing them ten times with chloroform:methanol (1:1, v:v).

         Caution: Chloroform is a hazardous solvent. Conduct all work in a fume hood, while wearing proper protective clothing.

      2. To have a 5 mg/mL lipid film, transfer 500 μL of a 10 mg/mL DOPC stock solution (see Recipe 5) into a round bottom glass tube on ice using Hamilton syringes. For 5 mg/mL DOPC:DOPE or DOPE:DOPS lipid mix, add 9 mol DOPC and 1 mol DOPE or DOPS (lipid stocks, see Recipe 5). In this study, the lipid mixtures DOPC:DOPE and DOPC:DOPS will be named only DOPE and DOPS, respectively.

         Caution: Avoid any use of plastic ware when handling organic solvents. For more complex or different lipid mixtures, the volume of used lipids needs to be adjusted.

      3. Evaporate the organic solvent at room temperature under reduced pressure in a rotary evaporator at 250 mbar for 2–4 h followed by evaporation at ~10 mbar for 15 min (see Figure 2A and 2B).

      4. Store the lipid film at -20 °C until use.

      5. Dissolve the lipid film in 1 mL of chloroform:methanol (1:1; v:v).

      6. Transfer into a glass vial with screw caps closed with parafilm and store it at -20 °C.

   2. Cleaning of the glass slides

      1. Clean the glass slides with detergent, deionized water, and 70% ethanol.

      2. Dry the slides with wipes.

      3. Place glass slides in the UV/ozone cleaner.

      4. Run the UV/ozone cleaner for 30 min.

         Caution: The UV/ozone cleaner must be placed under a fume hood.

      5. Turn off the UV/ozone cleaner and wait at least 15 min before opening the chamber.

   3. Preparation of GUVs

      1. Glue the O-ring using vacuum grease onto the cleaned side of one of the glass slides to have a tight, closed chamber. Apply 35–40 drops of 1 μL of lipid mix inside the O-ring under the fume hood (see Figure 2C).

      2. Evaporate the solvent under vacuum for 30–60 min at 250 mbar in a desiccator.

      3. Fill the O-ring with swelling buffer (see Recipe 6) and place the other glass slide on top with the cleaned side facing downwards.

      4. Place the chamber in a dark room for 2–4 h at room temperature (see Figure 2D).

      5. Carefully tap on each side of the chamber.

      6. Transfer the GUVs to a 2 mL tube by removing the glass slide and O-ring.

      7. Store the GUVs covered in aluminum foil at room temperature.

         
         

      
      Figure 2. Generation of giant unilamellar vesicles (GUVs) from lipid mixtures. (A, B) Preparation of the lipid mixture. The desired volume and type of lipid is mixed in chloroform:methanol (1:1; v:v) in a glass tube and (I) the solvent is evaporated in rotary evaporator under the reduced pressure of 250 mbar for 2–4 h; (II) the resulting thin lipid film on the glass tube is dissolved in chloroform:methanol (1:1; v:v) and (III) stored in a glass vial with screw caps sealed with parafilm. (B) The glass tube containing the lipids in chloroform is connected to the rotary evaporator. (C) Equipment for a GUV formation of the home-made chamber. (D) Schematic workflow of GUV formation by the swelling method. The lipid mixture is applied to the cleaned glass slides and (I) dehydrated in a desiccator at 250 mbar for 30–60 min. The dried lipid film is then (II) rehydrated in a swelling buffer for 2–4 h, resulting in the formation of giant vesicles.

      
      

   4. Annexin V staining of GUVs

      1. Place 25 μL of GUVs (with a cut tip) in 25 μL of binding buffer (see Recipe 7) on a cover glass slide.

         Note: As additional control, a GUV sample in buffer without Ca²⁺ can be prepared.

      2. Place the cover glass slide under the microscope.

      3. Let the GUVs settle for approximately 5 min before imaging in a white light channel and an annexin V channel.

Data analysis

Data acquisition

Images are acquired at a Leica TCS SP8 confocal laser scanning microscope (Leitz, Wetzlar, Germany) equipped with a 63×/1.20 water objective. For detailed imaging settings, see Table 1. All images are acquired at the same resolution, magnification, and orientation. This allows direct comparison of images and saves time when arranging figures.

Table 1. Confocal microscope settings

For analysis of the adherent cells

1. Export the raw image data in a format compatible with ImageJ (e.g., .tif) from the imaging system.

2. Open the ImageJ software and import the images of the blue fluorescence (DAPI) and the red fluorescence (Annexin V conjugated to Alexa Fluor 568).

3. Click on Image in the upper operation row of the interface and select in the color menu Merge Channels.

4. For C1 (red), the image showing the red fluorescence needs to be selected; for C3 (blue), the DAPI picture needs to be chosen. Press Ok to merge the two images.

5. Click on Image in the upper operation row of the interface and change the image type to RGB Color.

6. Save the images as a .tif or .jpg file by selecting the File menu in the upper operation row of the interface and press Save As. Representative images are shown in Figure 3.

   
   

   
   Figure 3. Representative confocal images of proliferating C2C12 wild-type and CDC50A knockout cells. Cells are stained with Alexa Fluor 568 conjugated annexin V (red) for 10 min in ice-cold TBSS with and without calcium. DAPI staining is used for visualization of nuclei. In contrast to C2C12 wild-type cells, CDC50A-deficient cells stained positive for annexin V, indicating increased surface exposure of aminophospholipids. Images are representative of three independent experiments. Scale bar: 30 μm.

For analysis of GUVs

ImageJ is used to analyze the membrane fluorescence intensity of individual GUVs before and after annexin V treatment. Only unilamellar giant vesicles are used for analysis.

1. Open the images with ImageJ by importing the LIF-file.

2. Split the channels to have bright light and annexin channel separately and save as Tiff.

3. Tiff images can be assembled (see Figure 4).

   
   

   
   Figure 4. Imaging the binding of fluorescent annexin V on giant unilamellar vesicles (GUVs). Giant unilamellar vesicles are prepared from different lipids and incubated without (-) and with (+) annexin V in the presence of Ca²⁺. Vesicles are observed in bright light and annexin channel. DOPC, PC (18:1/18:1) only; DOPE, PC (18:1/18:1)/PE (18:1/18:1), (9/1, mol/mol); DOPS, PC (18:1/18:1)/PS (18:1/18:1), (9/1, mol/mol). Data shown are from one experiment representative of two independent vesicle preparations. Scale bar: 10 μm.

Extract signal intensities with ImageJ software

1. Continue with the annexin channel.

2. A region of interest (ROI) is placed around the GUV (ROI_outer) of interest to measure the annexin V fluorescence intensity of the membrane, by measuring the integrated density value per pixel.

3. The second ROI is placed within the GUV lumen (ROI_inner) (see Figure 5A).

4. The inner ROI is subtracted from the outer ROI to have the membrane density value per pixel of the annexin V fluorescent intensity (ΔI):

   ΔI = I(ROI_outer) - I(ROI_inner)

5. Repeat the procedure for each GUV and save the data as an Excel (.xls) file.

6. The annexin V fluorescence intensities are plotted in form of a bar diagram using the OriginPro (see Figure 5B and Table 2).

   
   

   
   Figure 5. Fluorescence intensity analysis on giant unilamellar vesicles (GUVs). (A) A first region of interest (ROI) is placed around the GUV (ROI_outer), and a second ROI is placed within the GUV lumen (ROI_inner) of interest to measure the annexin V fluorescence intensity of the membrane, by measuring the integrated density value per pixel using the software ImageJ. (B) The mean annexin V fluorescence intensities for GUVs with the indicated lipid compositions are presented as averages, based on individual measurements of n ≤ 10 GUVs. Error bars indicate standard deviations. Data are from one experiment representative of two independent vesicle preparations.

   
   

   Table 2. The mean annexin V fluorescence intensities (ΔI) for giant unilamellar vesicles (GUVs) with the indicated lipid compositions. Data are from one experiment representative of two independent vesicle preparations. Data are plotted in Figure 5.
   

   Lipids of GUVs	-Annexin V		+Annexin V
   	ΔI, average (n = 10)	s.d.	ΔI, average (n = 13–15)	s.d.
   DOPC	0.001	0.001	1.430	1.814
   DOPE	0.086	0.114	1.107	1.229
   DOPS	0.001	0.002	16.732	8.934

Recipes

Buffers were prepared using double-distilled water (ddH₂O), which was obtained using an in-house water distillation system. Alternatively, all buffers are prepared using ultrapure water with purification sensitivity of 18 MΩ·cm^-1 at 25 °C.

1. Growth medium

   Open a 500 mL flask of high-glucose DMEM medium

   Add 100 mL of FBS (heat-inactivated)

   Optional: add 5 mL of 100× penicillin-streptomycin solution

   Prepare in sterile cabinet; store at 4 °C

   
   

2. TBSS buffer + Ca²⁺ (0.5 L)

   136 mM NaCl (3.97 g)

   2.6 mM KCl (96.9 mg)

   1.8 mM CaCl₂ (132.3 mg)

   1 mM MgCl₂·6H₂O (101.6 mg)

   0.36 mM NaH₂PO₄·2H₂O (24.8 mg)

   5.56 mM D-glucose (500.8 g)

   5 mM HEPES (600 mg)

   Adjust pH to 7.4 with 1 M NaOH. Complete volume to 0.5 L. Sterilize by filtering using a 0.22 μm filter. Store at 4 °C up to several months.

   
   

3. TBSS buffer - Ca²⁺ (0.5 L)

   136 mM NaCl (3.97 g)

   2.6 mM KCl (96.9 mg)

   1 mM MgCl₂·6H₂O (101.6 mg)

   0.36 mM NaH₂PO₄·2H₂O (24.8 mg)

   5.56 mM D-glucose (500.8 g)

   5 mM HEPES (600 mg)

   100 μM EGTA (19 mg)

   Adjust pH to 7.4 with 1 M NaOH. Complete volume to 0.5 L. Sterilize by filtering using a 0.22 μm filter. Store at 4 °C up to several months.

   
   

4. DAPI stock solution (1 mg/mL)

   Dissolve DAPI in ultrapure water to 1 mg/mL. Stock solution is stable for several months and repeated use, if stored protected from light at -20 °C.

   
   

5. Lipid stocks in chloroform

   Lipids are ordered in chloroform at a concentration of 25 mg/mL and stored at -20 °C until further use. For longer storage, aliquot 10 mg of lipids in glass vials with screw caps, evaporate the chloroform, and store the dried lipid at -20 °C. Before using it, dissolve the 10 mg of lipid in 1 mL of chloroform:methanol (1:1; v:v).

   Critical: Some lipids may have limited or very poor solubility in chloroform:methanol (1:1; v:v) and require a mixture of chloroform:methanol:water.

   
   

6. Swelling buffer (320 mM)

   Dissolve 5.48 g of sucrose to a final volume of 50 mL in deionized water. The buffer is filter-sterilized over a 0.2 μm Acrodisc^® syringe filter and stored at 5 °C.

   
   

7. Binding buffer (100 mL)

   10 mM HEPES-KOH pH 7.4 (238.31 mg)

   150 mM NaCl (876.6 mg)

   5 mM KCl (37.275 mg)

   1 mM MgCl₂·6H₂O (20.33 mg)

   1 mM CaCl₂ (11.098 mg)

   Adjust pH to 7.4 with 1 M NaOH. Complete volume to 100 mL. Store at 4 °C up to several months.

Acknowledgments

We gratefully acknowledge Michelle Werner for technical assistance. This protocol was adapted from our previous work (Weingärtner et al., 2012; Grifell-Junyent et al., 2022; Herrera et al., 2022). The work was supported by the Lundbeckfonden (R221-2016-1005 to T.G.P.) and an instrument grant from the Deutsche Forschungsgemeinschaft (INST 213/886-1 FUGG to T.G.P.). HDU is a scholar of the Friedrich Ebert Foundation.

Competing interests

The authors declare that no competing interests exist.

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