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. esuspend 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

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, DRAQ7™ single control, and FMO CellTrace™ Violet control for compensation setup. CellTrace™ 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.