Key words

1 Introduction

Perforin-mediated cytotoxicity is a key pathway used by human CTL to annihilate their target cells. Within minutes or seconds after productive TCR engagement, the pore-forming protein perforin, granzyme A and B and other enzymes stored in CTL cytoplasmic granules (named lytic granules) are secreted at the CTL/target cell lytic synapse [1,2,3,4,5,6]. Perforin-mediated penetration of granzyme A and B into target cells triggers a complex cascade of apoptotic and pyroptotic pathways leading to target cell death [4, 7].

Recent observations revealed that target cells do not passively receive CTL-derived lytic components at the cell-cell contact site; on the contrary, they deploy rapid and efficient synaptic defense mechanisms to counteract CTL attack. We showed that melanoma cells are resistant to CTL-mediated cytotoxicity when compared to conventional cytotoxicity-sensitive target cells, thanks to a process of membrane reparation based on rapid lysosome exocytosis [2, 8]. The reparation process is triggered by perforin pore formation into target cell membrane and Ca2+ entry at the lytic synapse [3]. Lysosome exposure limits the efficacy of perforin-mediated cytotoxicity by inducing perforin degradation and rapid plasma membrane reparative turnover [2, 3]. Additional lines of evidence strengthened the notion of target cell resistance at the lytic synapse. Synaptic actin cytoskeleton polymerization in human breast cancer cells has been shown to limit NK cell-mediated cytotoxicity [9, 10]. The ESCRT-dependent repair mechanism has been identified as a key molecular machinery involved in membrane reparation in mouse cancer cells [11].

Lipophilic dies, such as FM1-43 and FM4-64, have been thoroughly used to monitor membrane turnover associated with cellular endo/exocytosis processes [12, 13] (Fig. 1). We showed that they can be used to visualize by time-lapse-microscopy, ongoing membrane recycling occurring at the lytic synapse during CTL-mediated cytotoxicity [3] and to quantify in a high through-put manner CTL and target cell membrane turnover by flow cytometry ([3]; L. Filali et al. filed patent WO2020109355).

Fig. 1
4 sets of schematics represent the F M lipophilic dye uptake in the plasma membrane. It depicts the medium, active dye uptake, and cell membrane regions in unbound F M dye nonfluorescent in the left panel, bound F M dye fluorescent and internalized F M dye fluorescent in the center panels, and internalized F M dye fluorescent dye in the right panel.

Schematic representation of FM lipophilic dye uptake in plasma membrane upon secretive/reparation turnover. Non-fluorescent lipophilic dye (pink) stably intercalates into the lipid bilayer increasing in fluorescence intensity (red)

In the present chapter, we describe this method and its use to monitor, at the same time: (i) the membrane re-modelling occurring in CTL upon TCR engagement; (ii) the reparative turnover occurring in target cells upon CTL attack.

2 Materials

  1. 1.

    HLA-A2-restricted human CD8+ CTL clones specific for the NLVPMVATV peptide of the cytomegalovirus protein pp65. CAUTION-human cell lines should always be treated as a biohazard following local regulations in approved facilities and with all associated precautions.

  2. 2.

    Clone medium: RPMI 1640 GlutaMAX medium supplemented with heat inactivated 5% human AB serum, 50 μM 2-mercaptoethanol, 10 mM Hepes, 1× MEM-Non-Essential Amino Acids Solution (Gibco or equivalent), 1× sodium pyruvate (Sigma-Aldrich or equivalent), 10 μg/mL ciprofloxacin (AppliChem or equivalent), 100 IU/mL human recombinant interleukin-2, and 50 ng/mL human recombinant interleukin-15 (Miltenyi Biotec or equivalent).

  3. 3.

    Human Peripheral Blood obtained and processed following ethical procedures and with approval of local regulatory authorities. CAUTION- human blood products should always be treated as a biohazard following local regulations in approved facilities and with all associated precautions.

  4. 4.

    Ficoll®Paque Plus, Cytiva 17-1440-03 (GE17-1440-03, Sigma or equivalent).

  5. 5.

    Phosphate buffered saline without calcium and magnesium (PBS), (GIBCO or equivalent).

  6. 6.

    Sterile 15 mL or 50 mL polypropylene centrifuge tubes with screw caps.

  7. 7.

    Centrifuge with a swinging bucket rotor.

  8. 8.

    JY EBV-transformed B cell line (ATCC 77441 or equivalent).

  9. 9.

    D10 cell line (isolated from metastatic melanoma patients, kindly provided by Dr. G. Spagnoli, Basel, Switzerland, [2]).

  10. 10.

    Complete medium: RPMI 1640 GlutaMAX medium supplemented with heat inactivated 10% fetal calf serum and 50 μM 2-mercaptoethanol, 10 mM Hepes, 1× MEM-NEAA (Gibco or equivalent), 1× sodium pyruvate (Sigma-Aldrich or equivalent), and 10 μg/mL ciprofloxacin (AppliChem or equivalent).

  11. 11.

    MycoAlert mycoplasma detection kit (Lonza or equivalent).

  12. 12.

    Oregon Green™ 488 Taxol, Bis-Acetate (Tubulin Tracker™ Green, Invitrogen T34075 or equivalent).

  13. 13.

    1 mM PKH67 in ethanol (Sigma PKH67GL or equivalent).

  14. 14.

    N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl) Hexatrienyl) Pyridinium Dibromide, (FM™4-64 Dye, Invitrogen, T13320 or equivalent).

  15. 15.

    15-well chambered μ-slide (Ibidi, Biovalley, 81506 or equivalent).

  16. 16.

    0.5 mg/mL Poly-D-Lysine hydrobromide in sterile tissue culture grade water (Sigma P6407 or equivalent).

  17. 17.

    Antigenic peptide: NLVPMVATV (CMV peptide p65 NV-9, GeneCust, Luxembourg or equivalent).

  18. 18.

    R5: RPMI-1640, 5% FCS, 1 mM HEPES.

  19. 19.

    Automated inverted microscope adapted for live cell time-lapse fluorescence microscopy with a temperature-controlled chamber maintained at 37 °C at constant 5% CO2 (see Note 1).

  20. 20.

    Computer workstation running ImageJ software.

  21. 21.

    Ethylenediaminetetraacetic disodium salt solution (EDTA), (Sigma 2854 or equivalent).

  22. 22.

    Sodium azide (Sigma-Aldrich, S2002 or equivalent).

  23. 23.

    N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) Pyridinium Dibromide, (FM™ 1-43 Dye Invitrogen, T3163 or equivalent).

  24. 24.

    eBioscience™ Fixable Viability Dye eFluor™ 780, (Invitrogen, 65-0865-14 or equivalent).

  25. 25.

    CellTrace™ Violet Cell Proliferation Kit, for flow cytometry, (Invitrogen C34557 or equivalent).

  26. 26.

    96-u-bottom tissue culture plates, (FALCON/CORNING, 353077 or equivalent) 24 well tissue culture plates (FALCON/CORNING, 353047 or equivalent).

  27. 27.

    FACS-Buffer: PBS, 1% heat inactivated foetal calf serum, 1% heat inactivated human serum, 0.1% sodium azide, 0.5 mM EDTA.

  28. 28.

    Flow cytometer (see Note 2).

3 Methods

3.1 Cell Culture

  1. 1.

    Dilute heparin anticoagulated human blood from healthy donors 1:1 with RPMI (see Note 3).

  2. 2.

    Put 15 mL of Ficoll®Paque Plus solution into a 50 mL centrifuge tube and carefully overlay with up to 30 mL of diluted blood without disturbing the interface.

  3. 3.

    Centrifuge at 800 × g in a well-balanced swinging bucket rotor for 30 min with no acceleration and no break.

  4. 4.

    Carefully collect the cloudy layer of cells at the interface between the plasma and the Ficoll-Paque with a 5 mL pipette (= peripheral blood mononuclear cells or PBMC) and transfer to a fresh 50 mL centrifuge tube. Fill with RPMI and pellet cells at 400 × g for 15 min.

  5. 5.

    Discard supernatant, resuspend pelleted PBMC and fill tube with fresh sterile RPMI.

  6. 6.

    Centrifuge at 400 × g 10 min.

  7. 7.

    Resuspend PBMC in 30 mL RPMI, take a sample for counting, centrifuge 180 × g for 10 min, discard supernatant and resuspend pellet to 1 × 106/mL in Clone medium.

  8. 8.

    Irradiate the PBMC at 35 Gy. (CAUTION- Use X-ray or γ-radiation with appropriate regulatory approval and safety procedures, see Note 4).

  9. 9.

    Stimulate CD8+ T cell clones Clone medium containing 1 μg/mL phytohemagglutinin with 1 × 106/mL irradiated PBMC. Use 24 well culture plates (2 mL final volume).

  10. 10.

    Do not touch the cultures for the first 3 days at least.

  11. 11.

    The irradiated PBMC will die after 2–3 days.

  12. 12.

    Maintain the viable CD8+ T cell clone cells at 1–2 × 106 per mL in Clone medium. Always use 24 well plates (or smaller if needed).

  13. 13.

    Restimulation of clones is routinely performed every 2–3 weeks. OK

3.2 Procedure for Time-Lapse Microscopy

  1. 1.

    Pulse target cells with 10 μM antigenic peptide for 2 h at 37 °C/5% CO2 in R5 and vortex tubes every 30 min (see Notes 5–7).

  2. 2.

    Wash the Ibidi chambers twice with sterile PBS. Add 40 μL of 0.05 mg/mL Poly-D-lysine (freshly diluted 1:10 v/v with sterile PBS) per well and leave for 10 min at 37 °C/5% CO2.

  3. 3.

    Flip the slide over and let it air dry for at least 10 min in the sterile tissue culture hood.

  4. 4.

    Rinse coated wells twice with sterile PBS, once with RPMI 1640 and once with R5 prior to use (see Note 8).

  5. 5.

    Stain CTL with 1 μM Tubulin Tracker™ Green (1:500, from stock solution in 10% Pluronic/90% DMSO) at 37 °C/5% CO2 in R5 for 30 min and vortex for a few seconds every 10 min. Alternatively, stain CTL with 2 μM PKH67 in R5 for 1–2 min at room temperature (see Notes 9–12).

  6. 6.

    Wash target cells and CTL in R5 3× at room temperature: spin 5 min at 400 × g twice. Wash once in R5 for 10 min at 180 × g (see Note 13).

  7. 7.

    Resuspend CTL at 80.000 cells/5 μL in R5.

  8. 8.

    Resuspend target cells at 20.000 cells/5 μL in R5.

  9. 9.

    At least 5 min before starting the imaging, mount the coated and washed ibidi chamber on the heated stage within the temperature-controlled chamber at 37 °C and constant 5% CO2 of the microscope and add 40 μL of R5.

  10. 10.

    Add the 5 μL of target cells (20.000 cells/well) into the preheated well (see Note 14).

  11. 11.

    Set acquisition conditions on your microscope: adjust laser powers and time of recording (usually 30 min) and choose 1–3 s interval between images.

  12. 12.

    Check by visual inspection, that target cells have sedimented on the bottom of the slide.

  13. 13.

    Delicately open the chambered slide and add FM4-64 at a final concentration of 1 μg/mL into the well with the target cells (note that the maximum volume per well is 50 μL).

  14. 14.

    Add 5 μL of the stained CTL (80.000 cells/well) to the well (see Note 14).

  15. 15.

    Adjust focus and start acquisition. Figure 2 and Electronic Supplementary Movie 1 show typical experiments of FM uptake as monitored by time-lapse live cell imaging.

Fig. 2
9 sets of images arranged in 3 rows and 3 columns. They depict the uptake of F M 4-64 at the contact sites between a melanoma cell and two cognate cytotoxic T lymphocytes at 02 : 24, 03 : 18, 04 : 09, 07 : 49, 08 : 41, 09 : 55, 10 : 54, 12 : 40 and 14 : 54.

Visualization of membrane turnover at the lytic synapse. Snapshots show FM4-64 (red) uptake at the contact sites between a melanoma cell and two cognate CTLs (loaded with PKH67, green) (see Note 15)

3.3 Procedure for Flow Cytometry

  1. 1.

    Pulse target cells with 10 μM antigenic peptide, or leave unpulsed, for 2 h at 37 °C/5% CO2 in R5. Vortex for a few seconds tubes every 30 min (see Notes 15–18).

  2. 2.

    Stain CTL with Cell Trace Violet (CTV, 1:1000, dilution according to manufacturer’s instructions, final concentration 10 μM) for 20 min at 37 °C/5% CO2 in R5. This staining allows to discriminate T cells from target cells in the gating strategy.

  3. 3.

    Wash target cells and CTL three times in R5: spin 5 min at 400 × g three times.

  4. 4.

    Resuspend both pulsed and unplused target cells at 100.000 cells/25 μL in R5.

  5. 5.

    Distribute 25 μL of target cells (20.000 cells) into the wells of a 96-u-bottom plate for conjugation with the T cells (use one plate per time point: 2, 5, or 15 min).

  6. 6.

    Prepare a solution of FM1-43 at 20 μg/mL in R5 (see Note 19).

  7. 7.

    Resuspend CTL at 40.000 cells/25 μL in R5 and add them to the target cells in the 96-u bottom wells (2 CTL:1 target cell ratio).

  8. 8.

    Add 50 μL of the FM1-43 solution to each well containing the target and the T cells in a total volume of 50 μL (25 μL + 25 μL) to obtain a final concentration of 10 μg/mL of FM1-43. Keep unstained cells as control for flow cytometry.

  9. 9.

    Pellet cells by centrifuging the 96-u-bottom plates for 1 min at 400 × g and incubate at 37 °C/5% CO2 for 2, 5, or 15 min (see Note 20).

  10. 10.

    At the end of the different incubation times, put the plates on ice.

  11. 11.

    Add 100 μL of ice-cold FACS buffer to each well and disrupt conjugates by pipetting cell pellets in each well up and down (use a multichannel pipet).

  12. 12.

    Spin plates for 2 min at 400 × g at 4 °C and discard supernatant.

  13. 13.

    Wash plates twice by adding 200 μL of ice cold FACS-Buffer/well: spin plates at 400 × g for 2 min at 4 °C (see Note 21).

  14. 14.

    Dilute the Fixable Viability Dye eFluor™ 780 1:1000 in ice cold FACS-Buffer (50 μL/well) (see Note 22).

  15. 15.

    After washing, add 50 μL of the diluted Fixable viability dye eFluor780 per well and incubate 20 min on ice and in the dark.

  16. 16.

    Wash plates twice with 200 μL ice cold FACS-Buffer/well: spin at 400 × g for 2 min at 4 °C.

  17. 17.

    Resuspend the cells in 100 μL ice cold FACS-Buffer (see Note 23).

  18. 18.

    Acquire samples on a Flow Cytometer (see Note 24). See Fig. 3 for the gating strategy.

  19. 19.

    Once the two cell populations are identified as target cells and T cells, it is possible to study the uptake of the fluorescent dye on each cell population individually by applying the analysis on the differently gated populations. The geometric mean of FM fluorescence intensity is measured and presented as histogram plots (Figs. 4a, c and 5a, c) (see Note 25).

  20. 20.

    Pooled data from different experiments are expressed as geometric means ± SEM (Figs. 4b, d and 5b, d) (see Note 25). A dose–response curve of FM1-43 uptake in CTL interacting for 2, 5, or 15 min with JY cells pulsed with increasing concentrations of the antigenic peptide is show in Fig. 6. Data show the high sensitivity of the method (see Note 26).

Fig. 3
A scatter plot of S S C-A: S S C-A versus F S C-A: F S C-A, marks the scattered dots for cell 69.0. It leads to a scatter plot of S S C-A: S S C-A versus e Fluor 780, it marks the dots scattered for alive cells 61.4. It further leads to a scatter plot of S S C-A: S S C-A versus C T V, it marks the scattered dots for targets 24.0 and T cells 65.1. The targets and T cells lead to 2 scatter plots that mark dots scattered for single cells 87.2 and 89.8 which results in 2 area graphs of count versus F M 1-43, respectively.

Gating strategy used for flow cytometry analysis. Left panels: Live cells were initially selected on side scatter/forward scatter criteria. Low staining fixable viability dye efluor-780, corresponding to live cells, were then selected. CTL were distinguished from target cells on the basis of their positivity for Cell Trace violet (CTV). Central panels: On each population we applied gates that allow excluding cell doublets. Right panels show histogram of FM1-43 fluorescence in the two cell populations

Fig. 4
Two area graphs and 2 line graphs. A and C, 2 area graphs of count versus F M 1-43. They plot areas for unstained, negative peptides, and positive peptides. The unstained area has the highest peak. B and D, 2 line graphs with error bars of F M 1-43 geometric mean versus time in minutes. They plot 3 lines for no C T L, target cells negative peptide, and target cells plus peptide in an increasing trend.

FM1-43 internalization is enhanced after CTL attack in resistant and sensitive target cells. Time kinetics of FM1-43 fluorescence intensity in D10 or JY cells unpulsed or pulsed with 10 μM antigenic peptide. Analysis was performed on target cells either alone or following conjugation with CTL during 2, 5, or 15 min. (a, c) Typical flow cytometry plots of FM1-43 fluorescence intensity on melanoma cells (a) or JY cells (c). (b, d) Plots show time dependent FM1-43 uptake in melanoma cells (b) and in JY cells (d). Geometric mean fluorescence intensities of samples are indicated. Data are from four independent experiments realized in duplicate. Two-way ANOVA test using GraphPad Prism software was used to determine the statistical significance after 15 min of conjugation. **P < 0.01; ***P < 0.001. Reproduced after modification from L. Filali et al. filed patent; WO2020109355, 2020 with permission from INSERMTransfert

Fig. 5
2 area graphs and 2 line graphs. A and C, 2 area graphs of count versus F M 1-43. They plot areas for unstained, negative peptides, and positive peptides. The unstained area has the highest peak. B and D, 2 line graphs with error bars of F M 1-43 geometric mean versus time in minutes. They plot 3 lines for C T L only, target cells negative peptide, and target cells plus peptide in an increasing trend.

FM1-43 internalization is equally enhanced in CTL after activation by resistant or sensitive target cells (a, c) Typical flow cytometry plots of FM1-43 fluorescence intensity on CTLs interacting with melanoma cells (a) or JY cells (c). (b, d) Time kinetics of FM1-43 fluorescence intensity in CTL either alone or following conjugation with D10 (b) or JY cells (d) during 2, 5, or 15 min. Geometric mean fluorescence intensities of samples are indicated. Data are from four independent experiments. Results are from the same experiments shown in Fig. 4. Two-way ANOVA test using GraphPad Prism software was used to determine the statistical significance after 15 min of conjugation. **P < 0.01; ***P < 0.001. (Reproduced after modification from L. Filali et al. filed patent; WO2020109355, 2020 with permission from INSERMTransfert)

Fig. 6
24 sets of typical flow cytometry plots in 3 panels. They plotted curves for 10 micrometers, 1 micrometer, 100, 10, 1, 0.1, 0.01 nanometers, and negative peptides, along with values of 27.9, 27.6, 22.9, 14.6, 3.90, 2.28, 2.14, and 2.30 percent for 2 minutes in the left panel, 53.1, 57.1, 51.8, 33.8, 17.1, 6.68, 2.79, and 2.16 percent for 5 minutes in the center panel, and 80.7, 78.3, 76.3, 60.8, 33.8, 25.2, 6.13, and 3.11 percent for 15 minutes in the right panel.

Typical flow cytometry plots showing dose response curves of FM1-43 uptake in CTL. FM1-43 uptake in CTL interacting for 2, 5, or 15 min with JY cells pulsed with increasing concentrations of the antigenic peptide is shown. The percentage of activated CTL is indicated. (Reproduced after modification from L. Filali et al. filed patent; WO2020109355, 2020 with permission from INSERMTransfert)

4 Notes

  1. 1.

    For example, we use an automated inverted microscope (Nikon) with a spinning-disk confocal scan head (Yokogawa) with a sCMOS Hamamatsu ORCA-Flash 4.0 V3 camera operated with MetaMorph software or a laser scanning confocal microscope (LSM780, Zeiss). On either system, we use a 63× objective (1.4 NA, oil immersion).

  2. 2.

    For example, we use a FACS MACSQuant 10 (Miltenyi Biotec) and analyze the resulting files using the FlowJo 10 software.

  3. 3.

    Ideally, the allogeneic PBMC should come from two or more donors to optimize performance of the feeder cells to promote growth of the CD8+ T cell clone.

  4. 4.

    The objective of the irradiation is to prevent T cells in the PBMC from dividing. If an appropriate radiation source is not available, 10 μg/mL mitomycin C for 1 h at 37 °C in complete medium followed by 5× washes can also be used to prevent division of feeder cells.

  5. 5.

    The combination of human HLA-A2–restricted clonal antigen specific CD8+ T cell clones with different HLA-A2+ antigen-presenting target cells, used in the above-described methods, is technically challenging and does not need to be the method of choice. It requires specialized cell culture and cloning skills which have been setup and adjusted over years of practice. It is possible to use polyclonal CD8+ T cell lines, purified from healthy donor blood samples using negative selection approaches and expanded with CD3/CD28 beads or CD3/CD28 antibodies coupled to tissue culture plates. Polyclonal T cells can be stimulated by conjugation with target cells pulsed with a cocktail of Bacterial Super Antigens (SAgs). SAgs bind directly to the T-cell receptor Vβ region and to the α1 domain of the MHC Class II molecules, bypassing the need for antigen processing and presentation [14]. More in general, this method can be applied to all cytotoxic lymphocytes (including anti-CD3 redirected target cell lysis, NK cell, CAR-T cells, etc.).

  6. 6.

    For successful live cell imaging it is important to make sure that cells are in optimal conditions (split target cells the day before the experiment, change medium, be gentle when suspending, avoid bubbles, etc.). Also, periodically check cell lines for mycoplasma using a sensitive test kit.

  7. 7.

    Perform experiments under sterile conditions. This will avoid bacterial contamination coming up during the time of imaging.

  8. 8.

    The choice of the chambered slides (shape, dimension, etc.) depends mainly on the type of mounted insert, on the heated stage of the microscope and on the number of available cells.

  9. 9.

    Staining with dyes, probes or antibodies depends a lot on the cell types (mouse, human, lines, primary cells, etc.). It is therefore important to assess the optimal concentrations, times of incubation, temperature, etc., in preparative experiments, to obtain the best results.

  10. 10.

    For experiments requiring cell fixation, it is possible to use a fixable FM analog (FM™ 1-43FX, fixable analog of FM™ 1-43 membrane stain, Invitrogen™ F35355).

  11. 11.

    Prepare all solutions and dye dilutions the same day of the experiment. The coated chambered slides can be kept at 4 °C for a few days.

  12. 12.

    During staining and prior to imaging, keep all the solutions and cells at 37 °C/5% CO2.

  13. 13.

    Cells should never be put on ice. This will slow down any cellular process involved in the interaction between T cells and target cells and signal transduction. It can bias the results of the experiment and also alter the localization of the loaded dyes.

  14. 14.

    The ready to use cells, loaded with the dyes, will metabolize dyes and lose staining over time (1–2 h if left at RT, best), quicker if put at 37 °C) and will form aggregates in the tube (not suitable for investigating single cell interactions). In the case several time-lapse acquisitions are planned, it is important to prepare sequential lots of freshly loaded cells.

  15. 15.

    Here we describe the method to visualize FM day uptake at the synapse using time-lapse imaging. The membrane turnover occurring at both sides of the lytic synapse can be quantified by manual or automated image analysis as described in Filali et al. [3].

  16. 16.

    Design your experiment in advance and calculate the number of cells needed to perform it. Make sure that cells are in good conditions.

  17. 17.

    Prepare cells for experiments under sterile conditions.

  18. 18.

    Keep R5 at 37 °C/5% CO2.

  19. 19.

    Prepare the right amount of FM1-43 solution in advance.

  20. 20.

    Form conjugates with cells that have been kept at 37 °C. Keep centrifuge at room temperature. Never spin at 4 °C before the co-culture.

  21. 21.

    Keep FACS-Buffer at 4 ° C (on ice).

  22. 22.

    Prepare the right amount of diluted Fixable Viability Dye eFluor™ 780, and keep it on ice.

    Any other way to distinguish dead cells from alive cells can be used (additional Viability Dye eFluor, 7-AAD, PI, etc.).

  23. 23.

    Stop incubation on ice and rapidly disrupt conjugates using ice-cold FACS-Buffer.

  24. 24.

    FACS data are analyzed using FlowJo 10 software, but any other software will do.

  25. 25.

    The results are represented here as the geomean values, but the percentage of activated CTLs can also be presented.

  26. 26.

    FM1-43 uptake exhibits an antigen dose response comparable to that exhibited by CD107a surface upregulation, a gold standard method for quantifying CTL lytic granule secretion [3]. Interestingly, FM uptake is a more rapid procedure, does not require staining with antibodies, and allows to detect in parallel target cell membrane turnover.