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How to Measure NK Cell Killing Activity with a Standardized Potency Assay

5 Parts 10 Materials Print

Natural killer (NK) cells play a central role in innate immunity by directly recognizing and eliminating tumor cells. For immunology researchers developing NK cell-based therapies, reliable and standardized methods to measure NK cell natural cytotoxicity are essential. Standardized protocols enable meaningful comparison across experiments, NK cell preparations, and tumor models, strengthening data reliability. This flow cytometry-based protocol offers a flexible framework for assessing NK cell killing activity by incorporating multiple tumor cell lines, such as K562, A549, and SKOV-3, and supporting NK cells derived from peripheral blood, cord blood, or pluripotent stem cells.

Freshly isolated peripheral blood NK cells from healthy donors are widely used in cytotoxicity assays to establish baseline NK cell function and serve as a standardized reference source of effector cells. Resting, cytokine-activated, and expanded NK cells each serve distinct roles as reference controls in cytotoxicity assays: resting NK cells define basal cytotoxic activity, activated NK cells demonstrate the upper range of killing potential, and expanded NK cells are commonly evaluated during adoptive cell therapy development to assess product quality and performance. Beyond peripheral blood-derived NK cells, NK cells can also be differentiated from CD34+ stem and progenitor cells from cord blood or from human pluripotent stem cells (hPSCs). The assays described in this protocol have been validated using NK cells from multiple sources, as detailed in the Materials section.

Flow cytometry is a common methodology used to assess NK cell potency against various tumor targets in vitro. Accordingly, this protocol integrates complementary apoptosis readouts to capture distinct stages of NK cell-mediated killing. Annexin V identifies cell death by binding to externalized phosphatidylserine, while the tetramethylrhodamine ethyl ester (TMRE) assay detects earlier loss of mitochondrial membrane potential. TMRE provides stable signal detection and reduces false negatives in adherent tumor cell lines, as it is not hindered by EDTA-based detachment. By integrating both markers, this protocol provides a robust and adaptable framework for measuring NK cell potency across diverse target cell types.

Materials

Tumor Target Cell Lines and Recommended Culture Media
  • Tumor target cells
    • K-562 cells* (ATCC; Catalog #CCL-243)
    • A549 cells* (ATCC; Catalog #CRM-CCL-185)
    • SKOV-3 cells* (ATCC; Catalog #HTB-77)
  • Fetal bovine serum (FBS) (HyCloneâ„¢ Cytiva: Catalog #SH30066.03)
  • Iscove’s Modified Dulbecco’s Medium (IMDM) for K562 (Catalog #36150)
  • RPMI 1640 medium for A549 (Catalog #36750)
  • McCoy’s 5A medium for SKOV-3 (Catalog #36350)
  • Corning T-75 flasks (e.g. Catalog #200-0500)
  • D-PBS (without Ca++ and Mg++) (Catalog #37350)
  • Trypsin-EDTA (0.25%) solution (Catalog #07901)
  • CryoStor® CS10 (Catalog #100-1061)
  • 50 mL (30 x 115 mm) conical tube (e.g. Catalog #38010)

*To validate the NK cell potency assays, the following tumor cell lines were utilized: K-562 (ATCC® CCL-243™), A549 (ATCC® CCL-185™), and SKOV-3 (ATCC® HTB-77™). These cell lines were used as target populations to demonstrate the efficacy of the NK cell potency protocols and do not constitute a component of the commercial product. Users are responsible for obtaining the necessary licenses and/or agreements for the use of these cell lines within their own facilities.

NK Cell Preparation Materials
  • NK cells
    • Isolated from blood following an enrichment strategy, i.e. EasySepâ„¢ Human NK Cell Isolation Kit (Catalog #17955)** and primed overnight with activating cytokines, i.e IL-15 (Catalog #78218) at a concentration of 5 ng/mL
    • Isolated from blood and cultured and expanded during several days (10 - 15 days) using ImmunoCultâ„¢ NK Cell Expansion Kit (Catalog #100-0711)
    • Differentiated from cord blood-derived CD34+ cells (Catalog #70008) using StemSpanâ„¢ NK Cell Generation Kit (Catalog #09960)†
    • Differentiated from human pluripotent stem cells (hPSCs) using STEMdiffâ„¢ NK Cell Kit (Catalog #100-0170)†
  • Culture cell media for NK cells, i.e. ImmunoCultâ„¢ NK Cell Base Medium (Catalog #100-0712)‡ if working with peripheral blood NK cells or StemSpanâ„¢ SFEM II (Catalog #09655) if working with cord blood-derived NK cells or hPSC-derived NK cells
  • Hemocytometer (Catalog #100-1181)
  • Trypan Blue (Catalog #07050)
  • Human Recombinant IL-15 (rhIL-15), ACF (Catalog #78218)

**Based on the NK cell frequency found in peripheral blood (5 - 20%) and the EasySepâ„¢ Human NK Cell Isolation Kit recovery rate (75 - 90%), one can expect to obtain 3.75 x 106 - 1.8 x107 total NK cells from 1 x 108 peripheral blood mononuclear cells (PBMCs). Depending on the tumor cells used, number of replicates, number of plates, and effector to target ratios, the required amount of NK cells can vary significantly. It is recommended to calculate NK cell numbers needed and process the appropriate volume of PBMCs.
†NK cells from both StemSpan™ and STEMdiff™ kits were harvested at Day 21 - 23 instead of at Day 28 as suggested in the kit protocols. Our data indicated that there was no difference in cytotoxicity against the tumor target cells used in this protocol when comparing Day 21 - 23 and Day 28 NK cells. We recommend customers to optimize the duration of NK cell generation based on the expression of activating receptors as well as the ligands expressed on their target cell lines of interest.
‡Also available as part of the ImmunoCult™ NK Cell Expansion Kit. Individual kit components can be purchased by choosing the desired format from the dropdown menu on the linked product page.

Flow Cytometry-Based Potency Assay Materials

‡Also available as part of the ImmunoCult™ 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

Part 1: Preparation of Target Cells

Select the tumor target cell line most relevant to your study. The following procedures outline culture and preparation conditions for three commonly used models, K562 (Chronic Myelogenous Leukemia or CML-derived cell line), A549 (lung adenocarcinoma-derived cell line), and SKOV-3 (ovarian adenocarcinoma-derived cell line), representing both suspension and adherent tumor cell types.

1.1 Maintenance of Tumor Target Cells

K562
Culture medium: 10% fetal bovine serum (FBS) in Iscove’s Modified Dulbecco’s Medium (IMDM)
Subculturing procedure: Set cultures at a density of 1 - 2 x 105 cells/mL. Use 10 - 15 mL of medium in a Corning T-75 cm2 flask. Medium should be renewed every 2 - 3 days.

A549
Culture medium: 10% FBS albumin in RPMI 1640 medium
Subculturing procedure: Set cultures at a density of 1 - 2 x 105 cells/mL. Do not exceed 5 x 105 cells/mL. Use 10 - 15 mL of medium in a Corning T-75 cm2 flask. Medium should be renewed every 1 - 2 days.

  1. Remove and discard culture medium.
  2. Briefly rinse the cell layer with D-PBS to remove all traces of serum that contains trypsin inhibitors.
  3. Add 5 mL of Trypsin-EDTA solution to the flask and incubate for 8 - 10 minutes at 37°C until the cell layer is dispersed.
    Note: To avoid clumping, do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach.
  4. Add 10 mL of growth medium and aspirate cells by gently pipetting.
  5. Add appropriate aliquots of the cell suspension to new T-75 cm2 flask.
  6. Incubate cultures at 37°C.

SKOV-3
Culture medium: 10% FBS albumin in McCoy’s 5A medium
Subculturing procedure: Set cultures at a density of 1 - 2 x 105 cells/mL. Use 10 - 15 mL of medium in a Corning T-75 cm2 flask. Medium should be renewed every 2 - 3 days.

  1. Remove and discard culture medium. Briefly rinse the cell layer with D-PBS to remove all traces of serum that contains trypsin inhibitors.
  2. Add 5 mL of Trypsin-EDTA solution to the flask and incubate for 8 - 10 minutes at 37°C until the cell layer is dispersed.
    Note: To avoid clumping, do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach.
  3. Add 10 mL of growth medium and aspirate cells by gently pipetting.
  4. Add appropriate aliquots of the cell suspension to new T-75 cm2 flasks.
  5. Incubate cultures at 37°C.

1.2 Harvesting, Labeling, and Seeding Tumor Target Cells (One Day Before Potency Assay)

  1. Harvest tumor target cells from the T-75 cm2 flask when confluency is around 80 - 90%.
    1. Cells in suspension (e.g. K-562): Harvest 10 - 15 mL of cells in suspension and transfer to a new 50 mL conical tube. Top up to 45 mL with recommended medium.
    2. Adherent cell lines (e.g. A549, SKOV-3): Aspirate and discard 10 - 15 mL of medium overlaying the cells. Wash twice with D-PBS and discard. Add 5 mL of pre-warmed Trypsin-EDTA (0.25%) solution on top of the cells and incubate for 8 - 10 minutes at 37°C until the cell layer is dispersed. Add 10 mL of recommended medium (containing 10% FBS) to neutralize trypsin after detaching the cells from the culture flask. Transfer the cells now in suspension to a new 50 mL conical tube. Top up to 45 mL with recommended medium.
  2. Centrifuge the target cells at 300 x g for 10 minutes. Decant the supernatant.
  3. Resuspend the cells in 30 - 45 mL D-PBS. Centrifuge the target cells at 300 x g for 10 minutes. Decant the supernatant.
  4. Resuspend the cell pellet in 1 - 2 mL of D-PBS. The cell concentration should be within the range of 1 x 106 - 5 x 106 cells/mL.
  5. Count the cells using trypan blue and a hemocytometer.
  6. Take an aliquot of 1 x 105 cells and dilute them in 1 mL of the recommended medium for the indicated cell line. These unlabeled cells will be used for single stain compensation controls.
  7. Prepare a solution of CellTrace™ Violet fluorescent dye at a final concentration of 5 μM using pre-warmed D-PBS.
  8. Take the amount of cells needed for labelling (at least double the amount).
  9. Centrifuge the target cells at 300 x g for 10 minutes. Decant the supernatant.
  10. Resuspend the cells in the CellTrace™ Violet fluorescent dye solution at 1 x 106 cells/mL and incubate for 20 minutes at 37°C (protected from light to avoid fading).
  11. Add 2-fold volume of chilled quenching solution consisting of D-PBS or basal medium with 10% FBS (v/v) and incubate the cells for 5 minutes, protected from light.
  12. Top up the 50 mL tube with the recommended medium for the cell line and centrifuge at 300 x g for 10 minutes.
  13. Decant the supernatant and resuspend the cell pellet in 1 - 2 mL of recommended medium for the cell line.
  14. Perform a cell count and dilute cells to 1 x 105 cells/mL with the recommended culture medium for each cell line.
  15. Dispense 100 μL of the cell suspension in duplicate or triplicate into the 96-well tissue culture plate to act as test samples or controls (see Table 1).
    Note: Cells in suspension (e.g. K-562) are seeded in a 96-well round-bottom microplate and adherent cells (e.g. A549 and SKOV-2) in a 96-well flat-bottom microplate.
  16. Incubate at 37°C until the following day.

Table 1. Suggested Plating Layout for Fluorescent Labeling of Tumor Target Cells Prior to the Potency Assay

1 2 3 4 5 6 7 8 9
A Labeled Targets
(test sample)		 Labeled Targets
(test sample)		 Labeled Targets
(test sample)		 Labeled Targets
(test sample)		 Labeled Targets
(test sample)		 Labeled Targets
(test sample)		 Labeled Targets
(test sample)		 Labeled Targets
(test sample)		 Labeled Targets
(test sample)
B Labeled Targets
(test sample)		 Labeled Targets
(test sample)		 Labeled Targets
(test sample)		 Labeled Targets
(test sample)		 Labeled Targets
(test sample)		 Labeled Targets
(test sample)		 Labeled Targets
(test sample)		 Labeled Targets
(test sample)		 Labeled Targets
(test sample)
C Labeled Targets
(test sample)		 Labeled Targets
(test sample)		 Labeled Targets
(test sample)		 Labeled Targets
(test sample)		 Labeled Targets
(test sample)		 Labeled Targets
(test sample)		 Labeled Targets
(test sample)		 Labeled Targets
(test sample)		 Labeled Targets
(test sample)
D Labeled Targets
(spontaneous death)		 Labeled Targets
(spontaneous death)		 Labeled Targets
(spontaneous death)
E Unlabeled Targets
(unstained control)		 Unlabeled Targets
(TMRE single control)		 Unlabeled Targets
(Annexin V APC single control)		 Unlabeled Targets
(DRAQ7â„¢ single control)		 Labeled Targets
(CellTraceâ„¢ Violet single control)		 Labeled Targets
(FMO TMRE control)		 Labeled Targets
(FMO Annexin V APC control)		 Labeled Targets
(FMO DRAQ7â„¢ control)		 Labeled Targets
(FMO CellTraceâ„¢ Violet control)

An example layout for plating tumor target cells, both fluorescently labeled and unlabeled, in a 96-well tissue culture plate. This plate layout accommodates test samples, which include triplicates of three NK cell populations—peripheral blood NK cells, cord blood-derived NK cells, and hPSC-derived NK cells—at three effector/target (E/T) ratios (see Table 2 for details on NK cell types and ratios).
Unlabeled tumor cells should be added to the wells labeled as unstained control, TMRE single control, Annexin V APC single control, DRAQ7â„¢ single control, and FMO CellTraceâ„¢ Violet control for compensation setup. CellTraceâ„¢ Violet-labeled tumor cells should be added to all remaining wells to act as test samples, spontaneous death controls, and additional compensation controls. In the schematic, red wells indicate test samples, blue wells indicate spontaneous target cell death controls, and green wells indicate other control conditions.
The plate layout may be adjusted based on assay requirements, including the number of test samples, E/T ratios, and replicates. We recommend using a separate plate for each tumor cell line when assessing multiple targets, particularly when combining suspension (e.g. K-562) and adherent (e.g. A549 or SKOV-3) tumor cell lines.

Part 2: Preparation of NK Cells

  1. Harvest the NK cell population of interest (peripheral blood NK cells, cytokine-primed or expanded, and/or NK cells generated from cord blood or hPSC-derived CD34+ cells) and transfer them to a 14 mL polystyrene conical tube. Centrifuge at 300 x g for 10 minutes.
  2. Decant the supernatant and resuspend the cells in fresh ImmunoCultâ„¢ NK Cell Base Medium or StemSpan SFEM II.
  3. Count cells using trypan blue and a hemocytometer. Calculate the absolute viable NK cell numbers by multiplying the total viable cell count obtained with the hemocytometer by the frequency of CD56+ cells in your cell suspension, determined by flow cytometry.
    Note: Guidance for hemocytometer-based cell counting is provided in this protocol. The frequency of CD56+ cells may be analyzed by flow cytometry either the day before the assay or at this step.
  4. Prepare NK cell suspensions based on viable CD56+ NK cell numbers at least at three effector/target (E/T) ratios.
    1. We recommend using E/T ratios of 3:1, 1:1, and 0.3:1 for tumor target cells that are susceptible to NK cell killing (e.g. K-562) and 10:1, 3:1, and 1:1 for “difficult-to-kill†tumor target cells (e.g. A549 or SKOV-3).
      Example:
      1. 10 to 1 E/T ratio: 100,000 NK cells to 10,000 tumor target cells.
      2. 3 to 1 E/T ratio: 30,000 NK cells to 10,000 tumor target cells.
      3. 1 to 1 E/T ratio:10,000 NK cells to 10,000 tumor target cells.
      4. 0.3 to 1 E/T ratio: 3000 NK cells to 10,000 tumor target cells.
    2. Because 100 μL of NK cells will be added to 100 μL of tumor target cells, prepare NK cell dilutions at the following concentrations to achieve the indicated E/T ratios:
      1. 10 to 1 E/T ratio: 100,000 NK cells/100 μL or [NK] = 1 x 106 cells/mL.
      2. 3 to 1 E/T ratio: 30,000 NK cells/100 μL or [NK] = 3 x 105 cells/mL.
      3. 1 to 1 E/T ratio: 10,000 NK cells/100 μL or [NK] = 1 x 105 cells/mL.
      4. 0.3 to 1 E/T ratio: 3000 NK cells/100 μL or [NK] = 3 x 104 cells/mL.
      3. Tip: Prepare NK cell dilutions for five additional wells at the 1 to 1 E/T ratio ([NK] = 1 x 105 cells/mL) for use in single stains and compensation controls. Peripheral blood NK cells, either freshly isolated or cryopreserved in CryoStor® CS10 and stimulated overnight with 5 ng/mL recombinant human IL-15 (rhIL-15), serve as reliable control cells in this assay. Alternatively, peripheral blood NK cells expanded using the ImmunoCult™ NK Cell Expansion Kit can also be used as controls.

Part 3: Setting Up the Co-Culture (Target Cells and NK Cells)

  1. Add 100 μL of the NK cell suspension prepared at the various concentrations to the pre-seeded target cells in the 96-well tissue culture plate to establish a concentration curve of E/T ratios. Make duplicates or triplicates for each test sample (See Table 2).
    1. Cells in suspension: Culture blood NK cells, hPSC-derived NK cells, or cord blood-derived NK cells against K-562 targets at E/T ratios of 3:1, 1:1, and 0.3:1
    2. Adherent cell lines: Culture blood NK cells, hPSC-derived NK cells, or cord blood-derived NK cells against A549 or SKOV-3 targets at E/T ratios of 10:1, 3:1, and 1:1
  2. Set up the pre-seeded CellTrace™ Violet-labeled cells as spontaneous death control wells in triplicate (See Table 2) by topping up with 100 μL of the NK cell medium of choice (ImmunoCult™ NK Cell Base Medium or StemSpan™ SFEM II) for a final volume of 200 μL.
  3. Set up the required compensation controls (See Table 2).
    1. Unstained control: Top up the 100 μL of pre-seeded unlabeled target cells with 100 μL of NK medium.
    2. TMRE single stain: Top up the 100 μL of pre-seeded unlabeled target cells with 100 μL of NK medium.
      Note: At the end of the assay, this well will be treated with TMRE to obtain the TMRE single control. For an optimal TMRE signal, the tumor target cells need to be viable, which is why no NK cells are added in this control.
    3. Annexin V, APC single stain: Into the 100 μL of pre-seeded unlabeled target cells, dispense 100 μL of control NK cells (i.e. blood) at a 1:1 E/T ratio.
      Note: At the end of the assay, this well will be stained with Annexin V, APC. Since NK cells will kill tumor cells, this control will serve as an Annexin V, APC single control.
    4. DRAQ7™ single stain: Into the 100 μL of pre-seeded unlabeled target cells, dispense 100 μL of control NK cells (i.e. blood) at a 1:1 E/T ratio.
      Note: At the end of the assay, this well will be stained with DRAQ7â„¢. Since NK cells will kill tumor cells, this control will serve as a DRAQ7â„¢ single control.
    5. CellTrace™ Violet single stain: Top up the 100 μL of pre-seeded CellTrace™ Violet-labeled target cells with 100 μL of NK medium
    6. FMO TMRE: Into the 100 μL of pre-seeded CellTrace™ Violet-labeled target cells, dispense 100 μL of control NK cells (i.e. blood) at a 1:1 E/T ratio.
      Note: At the end of the assay, this well will not be treated with TMRE but will be stained with DRAQ7â„¢ and Annexin V, APC to obtain the FMO TMRE control.
    7. FMO Annexin V, APC: Into the 100 μL of pre-seeded CellTrace™ Violet-labeled target cells, dispense 100 μL of control NK cells (i.e. blood) at a 1:1 E/T ratio.
      Note: At the end of the assay, this well will be treated with TMRE and stained only with DRAQ7â„¢ (without Annexin V, APC) to obtain the FMO Annexin V, APC control.
    8. FMO DRAQ7™: Into the 100 μL of pre-seeded CellTrace™ Violet-labeled target cells, dispense 100 μL of control NK cells (i.e. blood) at a 1:1 E/T ratio.
      Note: At the end of the assay, this well will be treated with TMRE and stained only with Annexin V, APC (without DRAQ7â„¢) to obtain the FMO DRAQ7â„¢ control.
    9. FMO CellTrace™ Violet: Into the 100 μL of pre-seeded unlabeled target cells, dispense 100 μL of control NK cells (i.e. blood) at a 1:1 E/T ratio.
      Note: At the end of the assay, this well will be treated with TMRE and stained with Annexin V, APC and DRAQ7â„¢ to obtain the FMO CellTraceâ„¢ Violet control.
  4. Incubate targets and NK cells at 37°C and 5% CO2 for 24 hours to induce target cell killing.

Table 2. Suggested Plating Layout for Co-Culture of NK Cells with Tumor Target Cells During the Potency Assay

1 2 3 4 5 6 7 8 9
A Labeled Targets
+ Blood NK
3 to 1
(test sample)		 Labeled Targets
+ Blood NK
3 to 1
(test sample)		 Labeled Targets
+ Blood NK
3 to 1
(test sample)		 Labeled Targets
+ Blood NK
1 to 1
(test sample)		 Labeled Targets
+ Blood NK
1 to 1
(test sample)		 Labeled Targets
+ Blood NK
1 to 1
(test sample)		 Labeled Targets
+ Blood NK
0.3 to 1
(test sample)		 Labeled Targets
+ Blood NK
0.3 to 1
(test sample)		 Labeled Targets
+ Blood NK
0.3 to 1
(test sample)
B Labeled Targets
+ hPSC-NK
3 to 1
(test sample)		 Labeled Targets
+ hPSC-NK
3 to 1
(test sample)		 Labeled Targets
+ hPSC-NK
3 to 1
(test sample)		 Labeled Targets
+ hPSC-NK
1 to 1
(test sample)		 Labeled Targets
+ hPSC-NK
1 to 1
(test sample)		 Labeled Targets
+ hPSC-NK
1 to 1
(test sample)		 Labeled Targets
+ hPSC-NK
0.3 to 1
(test sample)		 Labeled Targets
+ hPSC-NK
0.3 to 1
(test sample)		 Labeled Targets
+ hPSC-NK
0.3 to 1
(test sample)
C Labeled Targets
+ CB-NK
3 to 1
(test sample)		 Labeled Targets
+ CB-NK
3 to 1
(test sample)		 Labeled Targets
+ CB-NK
3 to 1
(test sample)		 Labeled Targets
+ CB-NK
1 to 1
(test sample)		 Labeled Targets
+ CB-NK
1 to 1
(test sample)		 Labeled Targets
+ CB-NK
1 to 1
(test sample)		 Labeled Targets
+ CB-NK
0.3 to 1
(test sample)		 Labeled Targets
+ CB-NK
0.3 to 1
(test sample)		 Labeled Targets
+ CB-NK
0.3 to 1
(test sample)
D Labeled Targets
(spontaneous death)		 Labeled Targets
(spontaneous death)		 Labeled Targets
(spontaneous death)
E Unlabeled Targets
(unstained control)		 Unlabeled Targets
(TMRE single control)		 Unlabeled Targets
+ Blood NK
1 to 1
(Annexin V, APC single control)		 Unlabeled Targets
+ Blood NK
1 to 1
(DRAQ7â„¢ single control)		 Labeled Targets
(CellTraceâ„¢ Violet single control)		 Labeled Targets
+ Blood NK
1 to 1
(FMO TMRE control)		 Labeled Targets
+ Blood NK
1 to 1
(FMO Annexin V APC control)		 Labeled Targets
+ Blood NK
1 to 1
(FMO DRAQ7â„¢ control)		 Unlabeled Targets
+ Blood NK
1 to 1
(FMO CellTraceâ„¢ Violet control)

An example layout for co-culture of NK cells with tumor target cells in a 96-well tissue culture plate to measure NK cell killing activity.
In the schematic, red wells indicate test samples, blue wells indicate spontaneous target cell death controls, and green wells indicate other control conditions.
The plate layout may be adjusted based on assay requirements, including the number of test samples, E/T ratios, and replicates. We recommend using a separate plate for each tumor cell line when assessing multiple targets, particularly when combining suspension (e.g. K-562) and adherent (e.g. A549 or SKOV-3) tumor cell lines.

Part 4: TMRE Treatment and Annexin V, APC and DRAQ7â„¢ Staining

  1. Prepare a 200X TMRE working solution (250 nM) by diluting a 50 μM TMRE stock in the NK cell culture medium of choice (ImmunoCult™ NK Cell Base Medium or StemSpan™ SFEM II).
    Note: The recommended final concentration of TMRE is between 50 - 100 nM, and should be optimized to the target cell line of interest. For the tumor cell lines described in this protocol, a final concentration of 50 nM is recommended. Because the final well volume is 200 μL and 50 μL of TMRE working solution is added per well, a 250 nM working solution results in a 5-fold dilution to the desired final concentration.
  2. Add 50 μL of your prediluted TMRE solution to each of the following wells and mix carefully up and down (See Table 2):
    1. All test samples
    2. Spontaneous death controls
    3. TMRE single stain control
    4. FMO Annexin V, APC control
    5. FMO DRAQ7â„¢ control
    6. FMO CellTraceâ„¢ Violet control
      Important: Do not add TMRE to the unstained control, the Annexin V, APC single control, the DRAQ7â„¢ single control, nor the FMO TMRE control.
  3. Incubate the TMRE dye solution with cells for 30 to 60 minutes at 37°C and 5% CO2 (protected from light to avoid phototoxicity).
  4. Harvest cells:
    1. Cells in suspension (e.g. K-562):
      1. Centrifuge the round-bottom 96-well plates at 500 x g for 3 minutes to pellet both the targets and the NK cells.
    2. Adherent cells (e.g. A549, SKOV-3):
      1. Transfer 250 μL of cells (including NK cells and dead/floating targets) to a new 96-well round-bottom microplate and centrifuge at 500 x g for 3 minutes. Keep this plate at room temperature until the second fraction of cells that are still attached in the 96-well flat-bottom microplate are ready to be combined.
      2. Wash the 96-well flat-bottom microplate where the live and adherent cells still remain by adding 150 μL of D-PBS. Let stand for 30 seconds and discard the supernatant.
      3. Treat the adherent cells with 50 μL/well of Trypsin-EDTA (0.25%) solution and incubate for 8 - 10 minutes at 37°C.
      4. Add 100 μL of medium containing 10% FBS to neutralize trypsin and pipette up and down. This is the second fraction of cells that will be combined with the first fraction.
      5. Discard the supernatant of the 96-well round-bottom microplate that was spun down in the first step.
      6. Transfer 150 μL of the detached cells now in suspension (second fraction), combining it with the first fraction of cells in the 96-well round-bottom microplate. Centrifuge again at 500 x g for 3 minutes.
  5. Carefully remove the supernatant.
  6. Add 150 μL of D-PBS and centrifuge again at 500 x g for 3 minutes.
  7. Repeat steps 5 and 6 and carefully decant supernatant again.
  8. Prepare the Annexin V, APC and DRAQ7™ staining solution by diluting both dyes in Annexin V binding buffer at 1:500 and 1:250, respectively. Prepare 100 μL/well for staining.
    1. Annexin V, APC is recommended to be used at 0.2 μL/well and DRAQ7™ at 0.4 μL/well.
  9. Add 80 μL/well of the staining solution (Annexin V + DRAQ7™) to the test samples and appropriate controls and mix well.
  10. Add 80 μL/well of the appropriate staining solution to the stain controls and mix well.
  11. Incubate at room temperature for 15 minutes.
  12. Acquire samples on a flow cytometer. Collect at least 3000 - 5000 fluorescently labeled target cell events by setting the acquisition or recording rule on the labeled target cell gate.
    Note: Adjust FSC and SSC parameters for the tumor target cells, ensuring the FSC/SSC gate includes all target cells, including both live and dead populations. CellTraceâ„¢ Violet and TMRE signals are usually very bright; detector gains or voltages may require adjustment to avoid signal saturation.

Part 5: Assessing Potency of NK Cells Against Tumor Target Cells

  1. Analyze the target cell killing by gating on the fluorescently labeled target cells. Assess the frequency of apoptotic cells by measuring the apoptotic and dead target cells by plotting DRAQ7â„¢ vs. TMRE and gating on TMRE-negative cells in all test samples.
    1. Excitation/emission of TMRE-PE: 552/574 nm (Yellow 550).
    2. Excitation/emission of CellTraceâ„¢ Violet: Violet 405/450nm (Violet 405).
      Note: The standard deviation between triplicates should always be lower than 10% for reliable analysis.
  2. Calculate the average TMRE-negative frequency in labeled spontaneous dead controls.
    Note: The average TMRE-negative frequency in labeled spontaneous dead controls should remain below 10%.
  3. Calculate the percentage of tumor killing using the average percentage of TMRE-negative target cells in the test sample:
    Example: % Tumor Killing = [Average %TMRE-negative cells, test sample]
    Note: Alternatively, tumor killing may be calculated using the percentage of Annexin V-positive target cells instead of TMRE-negative cells. Using peripheral blood NK cells, we have confirmed that TMRE-negative cells and Annexin V-positive readouts correlate in potency assays using K-562, A549, and SKOV-3 target cells, indicating that both readouts can be used interchangeably.

Results

Workflow diagram representing the potency assay protocol for hPSC-derived NK cells

Figure 1. Potency Assay Protocol for hPSC-Derived NK Cells

Peripheral blood natural killer (NK) cells were used as positive control for the assay. Fresh leukopaks were processed using EasySep™ Human NK Cell Isolation Kit and purified NK cells were stimulated overnight with 5 ng/mL of recombinant human IL-15 (rh IL-15). Human pluripotent stem cell (hPSC) lines H1, STiPS-F016, STiPS-M001, and SCTi004-A were differentiated to NK cells using the STEMdiff™ NK Cell Kit. Tumor cells from K562, SKOV-3, and A549 cell lines were harvested, labeled with CellTrace™ Violet fluorescent dye, and pre-seeded one day prior to the potency assay. Tumor cells were co-cultured with hPSC-derived NK cells or peripheral blood NK cells for 24 hours, with TMRE added at a final concentration of 50 nM for the last 30 minutes. Adherent tumor cell lines were treated with Trypsin-EDTA for 8 minutes at 37°C prior to staining for apoptosis. All cells were resuspended in Annexin V Binding Buffer containing Annexin V and DRAQ7™ before flow cytometry data was acquired for analysis.

Killing Potency of hPSC-Derived NK Cells Determined by Flow Cytometry

Figure 2. hPSC-Derived NK Cells Show Potency Against Multiple Target Cell Lines

Tumor cell lines K562, SKOV-3, and A549 were analyzed by flow cytometry after 24 hours of co-culture with peripheral blood natural killer (NK) cells or human pluripotent stem cell (hPSC)-derived NK cells at different effector/target (E/T) ratios, as described in Figure 1. Tumor cells were identified by calculating the percentage of cells within the CellTrace™ Violet-positive gate. Tumor killing efficiency (% Tumor Killing, y-axis) was calculated as the percentage of TMRE-negative cells within the CellTrace™ Violet-positive tumor cell gate. Data represent mean ± SEM (blood NK cells: n = 4 - 7; hPSC-derived NK cells: n = 9 - 13). Dotted lines indicate the average percentage of TMRE-negative cells in spontaneous death controls (spon.death), pooled across experiments (n = 13). Two-way Anova tests were performed comparing blood NK cells and NK cells derived from hPSC lines: H1 (H1-NK), STiPS-F016 (F016-NK), STiPS-M001 (M001-NK) and SCTi004-A (4A-NK). A significant difference was observed for A549 targets (p < 0.0001 and SKOV-3 (p = 0.0172), but not for K562 (p = 0.5471). hPSC-derived NK cell killing activity is comparable to that of blood NK cells against K562 cells and is higher against A549 and SKOV-3 cells.

Workflow diagram representing the potency assay protocol for cord blood-derived NK cells

Figure 3. Potency Assay Protocol for Cord Blood-Derived NK Cells

Peripheral blood natural killer (NK) cells were used as positive control for the assay. Fresh leukopaks were processed using EasySep™ Human NK Cell Isolation Kit and purified NK cells were stimulated overnight with 5 ng/mL of recombinant human IL-15 (rh IL-15). Cord blood (CB)-derived NK cells were generated from frozen CD34+ cells using the StemSpan™ NK Cell Generation Kit. Tumor cells from K562, SKOV-3, and A549 cell lines were harvested, labeled with CellTrace™ Violet fluorescent dye, and pre-seeded one day prior to the potency assay. Tumor cells were co-cultured with CB-derived NK cells or peripheral blood NK cells for 24 hours, with TMRE added at a final concentration of 50 nM for the last 30 minutes of the assay. Adherent tumor cell lines were treated with Trypsin-EDTA for 8 minutes at 37°C prior to staining for apoptosis. All cells were resuspended in Annexin V Binding Buffer containing Annexin V and DRAQ7™ before flow cytometry data was acquired for analysis.

Killing Potency of CB-Derived NK Cells Determined by Flow Cytometry

Figure 4. CB-Derived NK Cells Show Potency Against Multiple Target Cell Lines

Tumor cell lines K562, SKOV-3, and A549 were analyzed by flow cytometry after 24 hours of co-culture with peripheral blood natural killer (NK) cells or cord blood (CB)-derived NK cells at the indicated effector/target (E/T) ratios, as described in Figure 3. Tumor killing efficiency (% Tumor Killing, y-axis) was calculated as the percentage of TMRE-negative cells within the CellTrace™ Violet-positive tumor cell gate. Data represent mean ± SEM (blood NK cells: n = 4 - 7; CB-derived NK cells: n = 5 - 7). Dotted lines indicate the average percentage of TMRE-negative cells in spontaneous death controls (spon.death), pooled across experiments (n = 7). Two-way Anova tests were performed comparing blood NK cells and NK cells derived from cord blood donors. A significant difference was observed for K562 targets (p = 0.0002) and A549 targets ( p = 0.0397), but not for SKOV-3 (p = 0.4889). CB-derived NK cell killing activity is lower than that of blood NK cells against K562, higher against A549 and comparable against SKOV-3 cells.

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