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How to Measure NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity Using Flow Cytometry-Based Assays

2 Options 15 Materials Print

Antibody-dependent cellular cytotoxicity (ADCC) is a key immune mechanism in which natural killer (NK) cells eliminate target cells whose surface antigens have bound specific antibodies. For immunology researchers and scientists developing NK cell- or antibody-based immunotherapies, having a reliable and standardized method to measure ADCC is essential. Standardized protocols help reduce variability, improve reproducibility across experiments and donors, and allow for consistent evaluation of NK cell potency and antibody efficiency. This protocol provides two different methods for measuring ADCC activity using flow cytometry.

The first method uses Annexin V and 7-AAD to identify apoptotic and dead NALM-6 target cells. The second employs TMRE, a mitochondrial membrane potential dye, to detect early apoptotic changes in adherent tumor cell lines such as A549 and SKBR3. These complementary methods allow researchers to quantify NK cell-mediated cytotoxicity with precision and flexibility, whether using suspension or adherent target cells. Each includes detailed steps for NK and target cell preparation, assay setup, staining, and data analysis to ensure robust and reproducible ADCC readouts.

Option 1: ADCC Protocol for Killing NALM-6 Target Cells Using an Annexin V Flow Cytometry-Based Assay

Materials

  • Human NK cells
  • NALM-6 cells
  • ImmunoCultâ„¢ NK Cell Base Medium (Catalog #100-0712*)
  • Phosphate-buffered saline (PBS)
  • Fluorescent dye i.e. CellTraceâ„¢ Violet (Thermo Fisher Scientific; Catalog #C34557) or eFluorâ„¢ 670 Cell Proliferation Dye (Thermo Fisher Scientific; Catalog #65-0840-85)
  • Fetal bovine serum (FBS)
  • Anti-CD19 monoclonal antibody i.e. Inebilizumab (Biorbyt; Catalog #orb1173649)
  • Annexin V Binding Buffer (Catalog #100-0334)
    • See More See Less

*Also available as part of the ImmunoCultâ„¢ NK Cell Expansion Kit. Individual kit components can be purchased by choosing the desired format from the dropdown menu on the linked product page.

Protocol

Preparation of NK Cells

  1. Harvest the NK cell population, transfer them to a 5 mL polystyrene round-bottom tube, and centrifuge at 300 x g for 10 minutes.
  2. Decant the supernatant and resuspend the cells in fresh ImmunoCultâ„¢ NK Cell Base Medium at a final concentration of 3 x 105 cells/mL.
  3. Prepare NK cell suspensions at various dilutions to generate a range of at least three effector/target (E/T) ratios.
    Example: 3 x 105 cells/mL, 1 x 105 cells/mL, and 0.3 x 105 cells/mL.

Preparation of Target Cells

  1. Harvest the NALM-6 cells and transfer them to a 50 mL polypropylene centrifuge tube. Keep a small aliquot of non-fluorescent target cells for single stain compensation controls.
  2. Add at least 5 fold volume of PBS and spin the sample at 300 x g for 10 minutes.
  3. Decant the supernatant and resuspend the cell pellet in 0.5 mL to 1 mL PBS. The cell concentration should be within the range of 5 x 106 - 1 x 107 cells/mL.
  4. Prepare a solution of fluorescent dye at 2X concentration.
    Example: Prepare a solution of CellTraceâ„¢ Violet or eBioscience Cell Proliferation dye eFluorâ„¢ 670 at 1/1000 dilution in PBS.
  5. Add dye solution at a 1:1 volume with the target cell suspension and incubate for 10 minutes at room temperature, protected from light.
  6. Add 4 fold volume of chilled quenching solution consisting of PBS or basal media with 10% FBS (v/v) and incubate the cells on ice for 5 minutes, protected from light.
  7. Top up the 50 mL tube with ImmunoCultâ„¢ NK Cell Base Medium and centrifuge at 300 x g for 10 minutes.
  8. Decant the supernatant and resuspend the cell pellet in 1 mL ImmunoCultâ„¢ NK Cell Base Medium.
  9. Split the cell suspension into two 5 mL polystyrene round-bottom tubes.
  10. Add 0.5 mL ImmunoCultâ„¢ NK Cell Base Medium to one tube. These target cells, without antibody, will be used as a negative control to monitor non-ADCC killing of target cells.
    Optional: An irrelevant biosimilar targeting an antigen that is not expressed on the surface of the target cells can also be used as a negative control if desired.
  11. To the other tube, add 0.5 mL of media supplemented with a 2X concentration of anti-CD19 monoclonal antibody. These targets will be used to monitor killing by ADCC.
    Example: Prepare a solution of Inebilizumab at 400 ng/mL in ImmunoCultâ„¢ NK Cell Base Medium. Transfer 0.5 mL of the biosimilar solution to 0.5 mL cells for a final concentration of 200 ng/mL.
  12. Incubate the anti-CD19 biosimilar monoclonal antibody with fluorescently labeled target cells for 30 minutes at room temperature, protected from light.
    Optional: Add 2 mL of ImmunoCultâ„¢ NK Cell Base Medium to both target cell suspensions and centrifuge at 300 x g for 10 minutes. Decant the supernatant and resuspend the target cells in 1 mL ImmunoCultâ„¢ NK Cell Base Medium.
  13. Count the target cells and prepare the target cell suspensions (both the negative and antibody-treated) at 1 x 105 cells/mL in ImmunoCultâ„¢ NK Cell Base Medium.
  14. Dispense 100 μL of target cells to round-bottom 96-well tissue culture plates to seed 1 x104 cells/well (see Table 1).
  15. Include a negative control with target cells that will not be co-cultured with NK cells. These are the “spontaneous†negative control samples (see Table 1).

Setting up the ADCC Plate

  1. Add 100 μL of the NK cell suspension prepared at the various concentrations to the target cells to establish a concentration curve of E/T ratios (see Table 1).
    Example: 3:1, 1:1, and 1:3.
  2. Set up additional compensation controls (see Table 1). The required compensation controls include:
    1. Unstained target cells
    2. Unstained target cells treated to generate Annexin V positive cells
    3. Unstained target cells treated to generate DRAQ7â„¢ or 7-AAD positive cells
    4. Fluorescently labeled target cells

Table 1. Suggested Plating Layout to Measure Killing of NALM-6 Target Cells Using Annexin V Flow Cytometry-Based Assay

E/T 3:1 1:1 1:3
1 2 3 4 5 6 7 8 9
A NK Sample 1 (S1) negative target S1 negative target S1 negative target S1 negative target S1 negative target S1 negative target S1 negative target S1 negative target S1 negative target
B NK Sample 1 (S1) ADCC target S1 ADCC target S1 ADCC target S1 ADCC target S1 ADCC target S1 ADCC target S1 ADCC target S1 ADCC target S1 ADCC target
C NK Sample 2 (S2) negative target S2 negative target S2 negative target S2 negative target S2 negative target S2 negative target S2 negative target S2 negative target S2 negative target
D NK Sample 2 (S2) ADCC target S2 ADCC target S2 ADCC target S2 ADCC target S2 ADCC target S2 ADCC target S2 ADCC target S2 ADCC target S2 ADCC target
E Spontaneous (spon) negative target Spon negative target Spon negative target Spon Antibody target Spon Antibody target Spon Antibody target
F Compensation (comp) control
No stain		 Comp control e450 Comp control PE-Ann V Comp control DRAQ7â„¢ or 7AAD

An example layout to set up for antibody-dependent cellular cytotoxicity (ADCC) staining and analysis using flow cytometry in a 96-well tissue culture plate.

Cells with light grey coloring signify that their corresponding wells should contain negative targets while dark grey coloring signifies ADCC targets, yellow coloring signifies spontaneous targets, and red coloring signifies various controls.

Staining the ADCC Samples

  1. Incubate targets and NK cells for 4 - 6 hours to induce target cell killing.
  2. Centrifuge the 96-well plates at 500 x g for 3 minutes.
  3. Decant 150 μL of supernatant from each well.
  4. Resuspend the cells in 150 μL ice-cold PBS.
  5. Centrifuge the 96-well plates at 500 x g for 3 minutes.
  6. Decant 150 μL of supernatant from each well.
  7. Prepare the Annexin V staining solution containing:
    1. 80 μL/well of Annexin V Binding Buffer,
    2. Add 0.15 μL/well PE-conjugated Annexin V
    3. Add 7-AAD dye at 1/250 dilution.
      Alternative: Add DRAQ7â„¢ at 1/2000 dilution
  8. Resuspend the PBS-washed cells in 80 μL/well of Annexin V staining solution. Incubate the cells with Annexin V solution for 20 minutes at room temperature, protected from light.
  9. Acquire samples on a flow cytometer. Collect at least 3000 fluorescently labeled NALM-6 target cell events.
  10. Analyze the target cell killing by gating on the fluorescently labeled target cells. Assess the frequency of apoptotic cells by measuring the Annexin V+/ 7-AAD- gate, or the dead target cells by measuring total Annexin V+ cell gate.
  11. Calculate the average Annexin V frequency in spontaneous negative controls.
  12. Subtract the spontaneous Annexin V levels from NK-containing samples.
    Example: [NK sample 1 @ 3:1 E/T] - [Average of spontaneous samples]
  13. Calculate the average Annexin V frequency of each NK-contained triplicate sample.
  14. Target cell killing by ADCC can be calculated by subtracting the negative target values from ADCC target values for each E/T ratio.
    Example: [Killing of NK sample 1 @ 3:1 E/T ratio with antibody] - [Killing of NK sample 1 @ 3:1 E/T ratio in the absence of antibody]
Gating Strategy of Annexin V-based Potency Assay

Figure 1. Gating Strategy of Annexin V-Based Potency Assay

Target cell death was measured by flow cytometry after gating on fluorescently labeled NALM-6 target cells (left panel) and monitoring the frequency of Annexin V+ target cells after co-culture with NK cells (right panel).

Antibody-Dependent Cell-Mediated Cytotoxicity Measured by an Annexin V-Based Killing Assay

Figure 2. Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Measured by an Annexin V-Based Killing Assay

NK cells isolated using the EasySepâ„¢ Human NK Cell Isolation Kit were co-cultured with NALM-6 target cells for 5 hours in ImmunoCultâ„¢ NK Cell Base Medium in the absence of antibody (negative) or in the presence of 250 ng/mL anti-CD19 antibody (ADCC). Data are shown as mean +/- SEM (n = 5 donors).


Option 2: ADCC Protocol for Killing A549 or SK-BR3 Target Cells Using a TMRE Flow Cytometry-Based Assay

Materials

  • Human NK cells
  • A549 or SK-BR3 cells
  • ImmunoCultâ„¢ NK Cell Base Medium (Catalog #100-0712*)
  • Trypsin-EDTA solution (Catalog #07091)
  • Phosphate-buffered saline (PBS)
  • Fluorescent dye i.e. CellTraceâ„¢ Violet (Thermo Fisher Scientific; Catalog #C34557) or eFluorâ„¢ 670 Cell Proliferation Dye (Thermo Fisher Scientific; Catalog #65-0840-85)
  • Fetal bovine serum (FBS)
  • Anti-EGFR monoclonal antibody i.e. Cetuximab Biosimilar (BioXCell; Catalog #SIM0002) or Anti-HER2 monoclonal antibody i.e. Trastuzumab Biosimilar (BioXCell; Catalog #SIM0005)
    • See More See Less

*Also available as part of the ImmunoCultâ„¢ NK Cell Expansion Kit. Individual kit components can be purchased by choosing the desired format from the dropdown menu on the linked product page.

Protocol

Preparation of NK Cells

  1. Harvest the NK cell population, transfer them to a 5 mL polystyrene round-bottom tube, and centrifuge at 300 x g for 10 minutes.
  2. Decant the supernatant and resuspend the cells in fresh ImmunoCultâ„¢ NK Cell Base Medium to generate a cell suspension appropriate for the target cell line of interest.
    Example: An NK cell suspension of 3 x 105 cells/mL may be appropriate for SK-BR3 target cells while an NK cell suspension of 1.5 x 106 cells/mL may be appropriate for A549 target cells.
  3. Prepare NK cell suspensions at various dilutions to generate a range of at least three E/T ratios.
    Example: 3 x 105 cells/mL, 1 x 105 cells/mL, and 0.3 x 105 cells/mL.

Preparation of Target Cells

  1. The day before setting up the ADCC assay, harvest adherent target cells from a T-75cm2 flask using the recommended protocol.
    Example: Trypsin-EDTA solution for 7 minutes, followed by the addition of the recommended medium for the cell line of interest to neutralize the trypsin.
  2. Centrifuge the target cells at 300 x g for 10 minutes. Decant the supernatant.
  3. Resuspend the cell pellet in 10 - 15 mL PBS. Centrifuge the target cells at 300 x g for 10 minutes then decant the supernatant.
  4. Resuspend the cell pellet in 0.5 - 1 mL PBS. The cell concentration should be within the range of 1 x 106 - 5 x 106 cells/mL.
  5. Remove 1 x 105 cells and dilute them in 1 mL of recommended medium for the cell line. These non-labeled cells will be used for single stain compensation controls at the end of the killing assay.
  6. Prepare a solution of fluorescent dye at 2X concentration.
    Example: Prepare a solution of CellTraceâ„¢ Violet or eBioscience Cell Proliferation dye eFluorâ„¢ 670 at 1/1000 dilution in PBS.
  7. Add dye solution at a 1:1 volume with the target cell suspension and incubate for 10 minutes at room temperature, protected from light.
  8. Add 4 fold volume of chilled quenching solution consisting of PBS or basal media with 10% FBS (v/v) and incubate the cells on ice for 5 minutes, protected from light.
  9. Top up the 50 mL tube with the recommended medium for the cell line and centrifuge at 300 x g for 10 minutes.
  10. Decant the supernatant and resuspend the cell pellet in 1 mL of the recommended medium for the cell line.
  11. Perform a cell count and dilute cells to 1 x 105 cells/mL.
  12. Dispense 100 μL of the cell suspension, both the fluorescently labeled and unstained (i.e. single stain compensation controls) and target cells into a flat-bottomed 96 well plate (see Table 2).
    Note: Take care to carefully add the target cells to the wells to form a uniform layer of cells. It is not recommended to dispense the cells with the pipette tip pointed at the middle of the well, as this can lead to the target cells becoming concentrated at the edges of the well and not forming a uniform monolayer.
  13. Culture the target cells for 12 - 18 hours in the recommended medium for the cell line. The target cells should attach and form a uniform layer of cells in the well.
  14. Approximately 30 minutes before co-culture with NK cells, prepare a solution of anti-EGFR or anti-HER2 monoclonal antibody at the appropriate concentration in ImmunoCultâ„¢ NK Cell Base Medium.
    Example: Prepare a solution of Cetuximab or InVivoSIM anti-human EGFR (Cetuximab Biosimilar) from 10 ng/mL to 1 μg/mL to find the ED50, or at the desired concentration. Alternatively, prepare a solution of Trastuzumab or InVivoSIM anti-human HER2 (Trastuzumab Biosimilar) from 10 ng/mL to 1 μg/mL to find the ED50, or at the desired concentration.
  15. Decant or flick off the recommended media for the cell line.
  16. Add 100 μL ImmunoCult™ NK Cell Base Medium to each well of control targets, without antibody. These samples will be used as a negative control to monitor non-ADCC killing of target cells (see Table 2).
    Optional: An irrelevant biosimilar targeting an antigen that is not expressed on the surface of the target cells can also be used as a negative control if desired.
  17. Add 100 μL ImmunoCult™ NK Cell Base Medium to each well of control targets containing the antibody of interest (see Table 2).
  18. Incubate the biosimilar monoclonal antibody with fluorescently-labeled target cells for 30 minutes at room temperature, protected from light.
  19. Decant or flick off the control or antibody-supplemented ImmunoCultâ„¢ NK Cell Base Medium.
  20. Add 100 μL of ImmunoCult™ NK Cell Base Medium to the target wells that will be used as the “spontaneous†negative control samples (see Table 2).
  21. The remaining cells will be co-cultured with NK cells, as described in the next section.

Setting up the ADCC Plate

  1. Add 100 μL of the NK cell suspension prepared at the various concentrations to the target cells to establish a concentration curve of E/T ratios (see Table 2).
    Example: 3:1, 1:1, and 1:3.
  2. Set up additional compensation controls (see Table 2). The required compensation controls include:
    1. Unstained target cells
    2. Unstained target cells treated to generate TMRE positive cells
    3. Unstained target cells treated to generate DRAQ7â„¢ or 7-AAD positive cells
    4. Fluorescently labeled target cells

Table 2. Suggested Plating Layout to Measure Killing of A549 or SK-BR3 Target Cells Using TMRE Flow Cytometry-Based Assay

E/T 3:1 1:1 1:3
1 2 3 4 5 6 7 8 9
A NK Sample 1 (S1) negative target S1 negative target S1 negative target S1 negative target S1 negative target S1 negative target S1 negative target S1 negative target S1 negative target
B NK Sample 1 (S1) ADCC target S1 ADCC target S1 ADCC target S1 ADCC target S1 ADCC target S1 ADCC target S1 ADCC target S1 ADCC target S1 ADCC target
C NK Sample 2 (S2) negative target S2 negative target S2 negative target S2 negative target S2 negative target S2 negative target S2 negative target S2 negative target S2 negative target
D NK Sample 2 (S2) ADCC target S2 ADCC target S2 ADCC target S2 ADCC target S2 ADCC target S2 ADCC target S2 ADCC target S2 ADCC target S2 ADCC target
E Spontaneous (spon) negative target Spon negative target Spon negative target Spon Antibody target Spon Antibody target Spon Antibody target
F Compensation (comp) control
No stain		 Comp control e450 Comp control TMRE Comp control DRAQ7â„¢ or 7AAD

An example layout to set up for antibody-dependent cellular cytotoxicity (ADCC) staining and analysis using flow cytometry in a 96-well tissue culture plate.

Cells with light grey coloring signify that their corresponding wells should contain negative targets while dark grey coloring signifies ADCC targets, yellow coloring signifies spontaneous targets, and red coloring signifies various controls.

Staining the ADCC Samples

  1. Incubate target cells and NK cells in an incubator, at 37oC and 5% CO2, for 4 - 20 hours to induce target cell killing.
  2. Prepare a 10X solution of TMRE in ImmunoCultâ„¢ NK Cell Base Medium.
    Note: The recommended final concentration of TMRE is 1 - 20 nM, and should be optimized to the target cell line of interest.
  3. Add 10 μL of the TMRE solution to the test samples containing NK cells, the “spontaneous†negative controls, and the single stain TMRE sample. Mix the solution and cells to ensure uniform labeling with the TMRE dye.
  4. Incubate the dye solution with cells for 30 - 60 minutes in the incubator at 37oC and 5% CO2.
  5. Collect dead and dying target cells by performing the following steps:
    1. Carefully remove all the liquid from the samples and transfer to a round-bottom 96-well tissue culture plate, maintaining the same layout (see Table 2).
      Tip: a multichannel pipette is recommended for the harvesting steps, described below.
    2. Add 100 μL of PBS to each well of the ADCC flat-bottom 96-well plate and gently mix.
    3. Decant the 100 μL of PBS from each well and add it to the corresponding sample from Step 5a in the round-bottom 96-well plate.
      Example: The liquid removed from position A1 in the plate in Step 5a and the PBS removed from position A1 in the plate in Step 5c will be combined together in position A1 of the new 96-well plate. This ensures that dying cells that have detached from the plate will be captured in final analysis of target cells.
    4. Centrifuge the round-bottom 96-well plate at 500 x g for 3 minutes.
    5. Carefully decant 150 μL of liquid from each well of the round-bottom 96-well plate, giving a final volume of approximately 50 μL.
  6. Collect the healthy and attached target cells by performing the following steps:
    1. Whilst the round-bottom 96-well plate is centrifuging, add 50 μL of trypsin-EDTA solution to the ADCC flat-bottom 96-well plate to remove healthy and attached target cells.
    2. Incubate at 37oC until the target cells have detached from the plate surface.
    3. Add 150 μL of solution containing 10% FBS to neutralize the trypsin.
    4. Mix thoroughly.
    5. Transfer the entire volume (approximately 200 μL) to the corresponding well of the round-bottom 96-well plate from Step 5. The final volume of each well will be approximately 250 μL.
      Example: The cells collected in position A1 in Step 5a & 5c and the cells from position A1 in Step 6 will be combined together. This ensures that dying cells that have detached from the plate and healthy cells that remained attached during the ADCC assay will be captured in final analysis of target cells.
  7. Spin the round-bottom 96-well plate at 500 x g for 3 minutes.
  8. Decant 200 μL from each well. Add 150 μL PBS or a FACs staining buffer. There should now be approximately 200 μL of volume in each well.
  9. Spin the round-bottom 96-well plate at 500 x g for 3 minutes.
  10. Decant 150 μL from each well. Add 150 μL PBS or a FACs staining buffer.
  11. Spin the round-bottom 96-well plate at 500 x g for 3 minutes.
  12. Decant 150 μL from each well.
  13. Prepare the DRAQ7â„¢ or 7-AAD staining solution in PBS or a FACs staining buffer:
    1. DRAQ7â„¢ at 1/2000 dilution
    2. 7-AAD dye at 1/250 dilution
  14. Add 100 μL of the DRAQ7™ or 7-AAD solution to each well and resuspend the pellet.
  15. Acquire samples on a flow cytometer. Collect at least 3000 fluorescently labeled target cell events.
  16. Analyze the target cell killing by gating on the fluorescently labeled target cells. Assess the frequency of apoptotic cells by measuring the TMRE low/ 7-AAD- gate, or the dead target cells by measuring total TMRE low cell gate.
    1. Excitation/Emission of TMRE: 552/574 nm
    2. Excitation/Emission of 7-AAD: 546/ 647 nm
  17. Calculate the average TMRElow frequency in spontaneous negative controls.
  18. Subtract the spontaneous TMRElow levels from NK-containing samples.
    Example: [NK sample 1 @ 3:1 E/T] - [Average of spontaneous samples]
  19. Calculate the average TMRElow frequency of each NK-contained triplicate sample.
  20. Target cell killing by ADCC mechanism can be calculated by subtracting the negative target values from ADCC target values for each E/T ratio.
    Example: [Killing of NK sample 1 @ 3/1 E/T ratio with antibody] - [Killing of NK sample 1 @ E/T ratio in the absence of antibody]
Gating Strategy of TMRE-Based Potency Assay

Figure 3. Gating Strategy of TMRE-Based Potency Assay

Target cell death was measured by flow cytometry after gating on fluorescently labeled A549 target cells (left panel) and monitoring the loss of TMRE signal in target cells after co-culture with NK cells (right panel).

Antibody-Dependent Cell-Mediated Cytotoxicity Measured by a TMRE-Based Killing Assay

Figure 4. Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Measured by a TMRE-Based Killing Assay

NK cells isolated using the NK cells isolated using the EasySep™ Human NK Cell Isolation Kit were co-cultured with A549 target cells for 20 hours in ImmunoCult™ NK Cell Base Medium in the absence of antibody (negative) or in the presence of 1 μg/mL anti-EGFR antibody (ADCC). Data are shown as mean +/- SEM (n = 6 donors).

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