A co-culture model system to quantify antibody-dependent cellular cytotoxicity in human breast cancer cells using an engineered natural killer cell line

Summary

Current protocols measure antibody-dependent cellular cytotoxicity (ADCC) in vitro using peripheral blood mononuclear cells (PBMCs), but isolation and variability among donors limit the viability and reproducibility of this approach. Here, we present a standardized co-culture model system to quantify ADCC on human breast cancer cells. We describe steps to engineer a natural killer cell line that stably expresses FCγRIIIa (CD16), required to mediate ADCC. We then detail the steps for the cancer-immune co-culture setup, followed by cytotoxicity measurement and analysis.

Graphical abstract

Highlights

• •A standardized co-culture system to perform scalable ADCC assays
• •Steps to engineer a natural killer cell line stably expressing FCγRIIIa (CD16)
• •Procedures to set up the co-culture system for cytotoxicity measurement and analysis

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Current protocols measure antibody-dependent cellular cytotoxicity (ADCC) in vitro using peripheral blood mononuclear cells (PBMCs), but isolation and variability among donors limit the viability and reproducibility of this approach. Here, we present a standardized co-culture model system to quantify ADCC on human breast cancer cells. We describe steps to engineer a natural killer cell line that stably expresses FCγRIIIa (CD16), required to mediate ADCC. We then detail the steps for the cancer-immune co-culture setup, followed by cytotoxicity measurement and analysis.

Before you begin

Institutional permissions

Primary human samples should be used in accordance with the appropriate ethical, safety and institutional regulations. For this study, donor material was obtained from the Sanquin blood bank, The Netherlands, subject to Sanquin’s ethical protocols.

Culturing of KHYG-1 (CD16+) cells

Timing: 30 min to 1 h

This step describes the maintenance of wild type KHYG-1 and KHYG-1 CD16+ cells. The KHYG-1 cell line is a natural killer cell line originating from the peripheral blood of a 45-year old female with aggressive leukemia.^1,2 These cells should be maintained at a density below 1∗10⁶ cells/mL and should be seeded with fresh KHYG-1 culture medium every 2–3 days. It is highly recommended to keep the passage number low. After thawing, allow the cells to recover for 2 weeks before performing cytotoxicity experiments. In contrast to other immortalized natural killer cell lines, the newly engineered KHYG-1 CD16+ cell line maintains FCγRIIIa (CD16) expression in culture (Figure 2B).

• 1.
  Heat inactivating 500 mL of FBS.

  • a.
    Preheat a waterbath, with enough water to cover an FBS bottle to above the level of FBS, to 56°C.
  • b.
    Thaw a 500 mL bottle of FBS in a 37°C waterbath.
  • c.
    Thoroughly mix the FBS.
  • d.
    Place the FBS in the 56°C waterbath.
  • e.
    Incubate the FBS for 30 min at 56°C.

    • i.
      Mix the FBS every 5–10 min.
  • f.
    Cool the FBS in an ice bath.
  • g.
    Aliquot the FBS in 50 mL tubes and store at −20°C. Thaw just before medium preparation.
• 2.
  Preparing KHYG-1 culture medium.

  • a.
    Add the following components to 435 mL RPMI 1640:

    • i.
      50 mL of heat inactivated FBS for final concentration of 10%.
    • ii.
      5 mL of 100 mM sodium pyruvate for a final concentration of 1 mM.
    • iii.
      5 mL of 200 mM L-glutamine for a final concentration of 2 mM.
    • iv.
      230 μL of 10 μg/mL IL-2 for a final concentration of 4.6 ng/mL.
    • v.
      5 mL of Penicilin (10,000 units/mL) – streptomycin (10 mg/mL) solution for a final concentration of 100 units/mL penicillin and 0.1 mg/mL streptomycin.
  • b.
    Mix gently by inverting.

CRITICAL: IL-2 is essential for growth and survival of KHYG-1 (CD16+) cells. Culture of KHYG-1 (CD16+) cells without IL-2 will result in reduced proliferation and cell death within 1 week.

• 3.
  Thawing of cryopreserved KHYG-1 (CD16+) cells.

  • a.
    Prepare 20% FBS KHYG-1 culture medium.
  • b.
    Thaw cells in a 37°C water bath. When most of the vial is thawed immediately transfer the cell suspension to 5 mL pre-warmed 20% FBS KHYG-1 culture medium to dilute the DMSO.
  • c.
    Centrifuge at 210 × g for 5 min at room temperature.
  • d.
    Remove supernatant and resuspend cells in 0.5 mL pre-warmed 20% FBS KHYG-1 culture medium.
  • e.
    Count cells using trypan blue to assess cell viability.
  • f.
    Seed cells at a density of 0.5∗10⁶ cells/mL in 20% FBS KHYG-1 culture medium in a 6-well plate.

Note: Cell proliferation can be irregular after thawing. The first week after thawing count cells daily and maintain at 0.2–1∗10⁶ cells/ml.

• 4.
  Passage KHYG-1 (CD16+) cells with fresh KHYG-1 culture medium every 2–3 days.

  • a.
    Gently resuspend the KHYG-1 (CD16+) suspension cells. Some cells may appear adherent on the bottom of the dish, but gentle resuspension or tapping will loosen them. Collect the cells in a 15 mL falcon.
  • b.
    Centrifuge at 210 × g for 5 min at room temperature.
  • c.
    Remove supernatant and resuspend cells in pre-warmed 10% FBS KHYG-1 culture medium.
  • d.
    Count cells using trypan blue to assess cell viability.
  • e.
    Seed cells at a cell density of 0.1–1∗10⁶ cells/mL in a 6-well plate or a T25 flask.
• 5.
  Cryopreservation of KHYG-1 (CD16+) cells.

  • a.
    Centrifuge at 210 × g for 5 min.
  • b.
    Remove supernatant and resuspend cells in pre-warmed 20% FBS KHYG-1 culture medium.
  • c.
    Count cells using trypan blue to assess cell viability.
  • d.
    Resuspend to 2∗10⁶ cells/mL in 20% FBS medium + 10% DMSO.
  • e.
    Immediately transfer 1 mL cell suspension to a cryovial. Place cryovials in a freezing container and the container in a −80°C freezer for minimum 16 h.
  • f.
    Transfer cryovials to liquid nitrogen for long term storage.

CRITICAL: Cell density should never exceed 1∗10⁶ cells/ml. Overconfluence of KHYG-1 CD16+ cells leads to reduced proliferation and loss of cytotoxicity.

Figure 2.

Generation of KHYG-1 cell line stably expressing FCγRIIIa (CD16)

(A) Schematic of CD16 overexpression construct for retroviral transduction.

(B) Expression of CD16 by KHYG-1 wild type and KHYG-1 CD16+ cell lines measured by flow cytometry before retroviral infection and at two different time points after transduction. KHYG-1 CD16+ cells stably express CD16 for over 3 months.

Culturing Phoenix amphotropic cells

Timing: 30 min

This step describes the maintenance of the Phoenix AMPHO cell line. This cell line is derived from human embryonic kidney HEK293T/17 cell line and expresses amphotropic envelope protein. It is a retrovirus packaging cell line used to create amphotropic retroviruses for transduction of human cell lines.

• 6.
  Coat cell culture dishes with gelatin.

  • a.
    Add 5 mL sterile 0.1% gelatin to a 10 cm culture dish.
  • b.
    Incubate 5–15 min.
  • c.
    Remove gelatin.
  • d.
    Open the lid slightly and allow the plate to dry for over 45 min.
  • e.
    Store plate at 20°C–25°C and use within 48 h.

Optional: Incubate plates with 0.1% gelatin for 16–24 h for optimal coating.

• 7.
  Thawing of Phoenix amphotropic cells.

  • a.
    Pre-warm 5 mL phoenix AMPHO culture medium in a 15 mL tube.
  • b.
    Thaw cells in a 37°C water bath. When most of the vial is thawed immediately transfer the cell suspension to 5 mL pre-warmed phoenix AMPHO culture medium to dilute the DMSO.
  • c.
    Centrifuge at 210 × g for 5 min.
  • d.
    Remove supernatant.
  • e.
    Resuspend the cell pellet in 10 mL pre-warmed phoenix AMPHO culture medium and transfer cells to a pre-coated 10 cm dish.
  • f.
    Place in a 37°C 5% CO₂ incubator.
• 8.
  Passage cells every 3–4 days when confluent.

  • a.
    Carefully wash cells with 20°C–25°C PBS.
  • b.
    Add 1 mL of 0.25% trypsin (2.5 g/L) to the cells and incubate for 1 min at 20°C–25°C.
  • c.
    Neutralize trypsin with pre-warmed phoenix AMPHO culture medium containing 10% FBS, resuspend and collect the cells in a 15 mL tube.
  • d.
    Centrifuge at 210 × g for 5 min.
  • e.
    Discard supernatant and resuspend the cell pellet in phoenix AMPHO culture medium.
  • f.
    Seed cells at a 1:10–1:20 dilution on a gelatin coated culture dish to ensure cell growth in subconfluent conditions.
  • g.
    Place in a 37°C 5% CO₂ incubator.
• 9.
  Cryopreserve cell lines.

  • a.
    Collect cells from an 80% confluent 15 cm dish using trypsin.
  • b.
    Centrifuge at 210 × g for 5 min.
  • c.
    Discard supernatant and resuspend the cell pellet in 5 mL phoenix AMPHO culture medium + 10% DMSO.
  • d.
    Immediately transfer 1 mL cell suspension to a cryovial. Place cryovials in a freezing container and the container in a −80°C freezer for minimum 16 h.
  • e.
    Transfer cryovials to liquid nitrogen for long term storage.

Note: Phoenix amphotropic cells do not adhere very well. Avoid shocks and gently change medium or wash the cells to avoid detaching the cells.

Culturing breast cancer cell lines BT474, MCF7 and MDA-MB-361

Timing: 30 min

This step describes the maintenance of BT474, MCF7 and MDA-MB-361 breast cancer cell lines. It is highly recommended to keep the passage number low. BT474 and MDA-MB-361 cell lines are HER2-overexpressing (HER2+) breast cancer cell lines. The MCF7 cell line expresses low levels of HER2 and is used as a control in the cytotoxicity experiments.

• 10.
  Thaw cryopreserved cell lines.

  • a.
    Pre-warm 5 mL culture medium in a 15 mL tube.
  • b.
    Thaw cells in a 37°C waterbath. When most of the vial is thawed immediately transfer cell suspension to 5 mL pre-warmed culture medium to dilute the DMSO.
  • c.
    Centrifuge at 210 × g for 5 min.
  • d.
    Remove supernatant.
  • e.
    Resuspend the cell pellet in 10 mL pre-warmed culture medium and transfer to a 10 cm dish.
  • f.
    Place in a 37°C 5% CO₂ incubator.
• 11.
  Passage cells every 3–4 days when confluent.

  • a.
    Wash cells with 20°C–25°C PBS.
  • b.
    Add 1 mL of trypsin and incubate the cells at 37°C for 3–5 min or until most cells detach by gently tapping the dish.
  • c.
    Neutralize trypsin with 3 mL of pre-warmed culture medium containing 10% FBS, resuspend and collect the cells.
  • d.
    Centrifuge at 210 × g for 5 min.
  • e.
    Discard supernatant and resuspend the cell pellet in culture medium.
  • f.
    Seed BT474, MCF7 and MDA-MB-361 at 1:4, 1:8, 1:3 dilutions respectively on a 10 cm dish to ensure cell growth in subconfluent conditions.
  • g.
    Place in a 37°C 5% CO₂ incubator.
• 12.
  Cryopreserve cell lines.

  • a.
    Collect cells from an 80% confluent 15 cm dish using trypsin.
  • b.
    Centrifuge at 210 × g for 5 min.
  • c.
    Discard supernatant and resuspend the cell pellet in 5 mL culture medium + 10% DMSO.
  • d.
    Immediately transfer 1 mL cell suspension to a cryovial. Place cryovials in a freezing container and place the container in a −80°C freezer for minimum 16 h.
  • e.
    Transfer cryovials to liquid nitrogen for long term storage.

Isolation of peripheral blood mononuclear cells (PBMCs) from whole blood

Timing: 4 h

This step describes the isolation of peripheral blood mononuclear cells (PBMCs) from whole blood taken from a healthy donor. In this study PBMCs were isolated from a buffy coat obtained from whole blood after centrifugation without a density gradient. Whole blood can also be used directly. This protocol requires a minimum of 20 mL of whole blood which results in about 2∗10⁷ PBMCs. These PBMCs were used to compare the cytotoxicity of the KHYG-1 CD16+ natural killer cell line. Natural killer cells make up about 5%–20% of the PBMCs.³

• 13.Add 10 mL Ficoll-Paque to a 50 mL tube. Centrifuge at 250 × g to collect all ficoll in the bottom.
• 14.Collect a buffy coat or collect 20 mL whole blood from a healthy donor using BD sodium heparin vacutainers.
• 15.Transfer the blood or 20 mL of buffy coat to a 50 mL tube and dilute 1:1 with room temperature PBS.
• 16.
  Very carefully layer the diluted blood on the ficoll.

  • a.
    Tilt the ficoll tube and very slowly pipet the blood against the top of the tube. The blood should flow against the wall and stay on top of the ficoll.
• 17.Centrifuge at 1200 × g for 20 min at 20°C–25°C at low acceleration (=1/9) and deceleration (=1/9). The blood is now separated in a clear yellow plasma layer on top, a white cloudy PBMC layer, a clear ficoll layer and in the bottom a red erythrocyte layer (Figure 1).
• 18.After the centrifugation, cool the centrifuge to 4°C.
• 19.Add 10 mL cold PBS to a new 50 mL tube.
• 20.Remove some of the plasma layer for easier access to the PBMCs. Use a 1000 μL pipet to take the PBMCs by moving on top of the PBMC layer in a circular motion.
• 21.Transfer the PBMCs to the tube containing 10 mL of cold PBS. Stop removing PBMCs when the erythrocyte layer starts moving.

Note: Any plasma and ficoll that is transferred with the PBMCs will be washed away in the next steps.

• 22.Add cold PBS to a total volume of 30 mL.
• 23.Centrifuge the PBMC suspension at 450 × g for 10 min at 4°C with low acceleration (=1/9) and intermediate deceleration (=3/9).
• 24.Remove the supernatant and resuspend the pellet in 10 mL cold PBS.

Note: the pellet should be white indicating the absence of red blood cells.

• 25.Separate a small fraction and count the PBMCs with trypan blue to assess cell viability.

Note: PBMC isolation should yield around 1∗10⁶ cells/ml of blood. Recovery (% of live cells) should be 95%–100%.

• 26.Centrifuge the PBMC suspension at 400 × g for 7 min at 4°C with low acceleration (=1/9) and intermediate deceleration (=3/9).
• 27.Remove the supernatant and resuspend the pellet in RPMI + 20% FBS + 1% PS + 10% DMSO to a cell density of at least 3∗10⁶ cells/mL.
• 28.Immediately transfer 1 mL cell suspension to a cryovial. Place cryovials in a freezing container and place the container in a −80°C freezer for minimum 16 h.
• 29.Transfer cryovials to liquid nitrogen for long term storage.
• 30.
  To thaw the PBMCs use the following steps:

  • a.
    Pre-warm 10 mL KHYG-1 culture medium in a 15 mL tube.
  • b.
    Thaw cells in a 37°C water bath. When most of the vial is thawed immediately transfer the cell suspension to 10 mL pre-warmed KHYG-1 culture medium to dilute the DMSO.
  • c.
    Centrifuge at 210 × g for 10 min.
  • d.
    Remove supernatant and resuspend the cell pellet in 2 mL pre-warmed KHYG-1 culture medium.
  • e.
    Count the cells and seed 1∗10⁶ cells/mL in a 6-well plate.
  • f.
    Allow cells to recover for 16–24 h in a 37°C 5% CO₂ incubator.

Figure 1.

Blood layers after ficoll separation

After centrifugation with ficoll, 4 layers can be observed in the blood: a clear yellow plasma layer, a white cloudy PBMC layer, a clear ficoll layer and a red erythrocyte layer.

Prepare cytotox 96 reagent

Timing: 15 min

• 31.
  Prepare assay buffer aliquots.

  • a.
    Thaw assay buffer of the Cytotox 96 kit (Promega).
  • b.
    Prepare aliquots with 12 mL of assay buffer in 15 mL falcon tubes.
  • c.
    Store aliquots at −20°C.
• 32.
  Combine substrate mix and assay buffer.

  • a.
    Thaw one 12 mL assay buffer aliquot to 20°C–25°C.
  • b.
    Equilibrate 1 vial of substrate mix to 20°C–25°C.
  • c.
    Add assay buffer to the substrate mix and mix gently until the substrate is dissolved.
  • d.
    Transfer to a 15 mL falcon tube, protect from light and keep on ice.
  • e.
    Use immediately for LDH measurement. Unused Cytotox 96 reagent can be stored at −20°C and used again.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Trastuzumab, anti-HER2	Pfizer	Trazimera
CD16-PE 3G8 (1:25)	BD Biosciences	Cat# 555407; RRID: AB_395807
igG-PE MOPC-21 (1:25)	BD Biosciences	Cat# 555749; RRID: AB_396091
Bacterial and virus strains
Amphotropic retrovirus	Generated in this study	N/A
Biological samples
Healthy adult PBMCs	Isolated from peripheral blood of a healthy donor	N/A
Chemicals, peptides, and recombinant proteins
Ficoll paque plus	Cytiva	17144002
Polybrene	Millipore	TR-1003-G
PBS (Dulbecco’s Phosphate buffered saline)	Sigma	D8537
Trypsin (0.25%), phenol red	Gibco	25050
DMEM, high glucose, pyruvate	Gibco	41966
Ham’s F10, stable glutamine	Pan Biotech	P04-13512
RPMI 1640 Medium, HEPES	Gibco	22400
Fetal bovine serum	Capricorn Scientific	FBS-12A
L-glutamine (200 mM)	Gibco	25030
Sodium pyruvate (100 mM)	Gibco	11360
MEM NEAA (100X)	Gibco	11140
Penicillin-Streptomycin	Sigma	P0781
Recombinant Human IL-2	PeproTech	Cat#200-02; Accession# P60568
Gelatin	Sigma	04055
BSA (bovine serum albumin)	Sigma	A3294
Lysis buffer	Promega	G182A
Lipofectamine 2000	Invitrogen	Cat# 11668-019
Puromycin	Sigma	P8833
Critical commercial assays
CytoTox 96 Non-radioactive Cytotoxicity Assay	Promega	G1780
Experimental models: Cell lines
Human: KHYG-1	DSMZ	Cat# ACC 725; RRID: CVCL_2976
Human: Phoenix-AMPHO	ATCC	CRL-3213; RRID:CVCL_H716
Human: KHYG-1 CD16+	Generated in this study	N/A
Human: BT474	ATCC	RRID:CVCL_0179
Human: MDA-MB-361	ATCC	RRID:CVCL_0620
Human: MCF7	ATCC	RRID:CVCL_0031
Recombinant DNA
Plasmid: pMSCV_IRES_GFP_CD16a	This study, Addgene	Cat# 196189
Other
BD Vacutainer plastic sodium heparin tube	BD Biosciences	Cat# 367876
15 cm tissue culture dish	Sarstedt	83.3903
10 cm tissue culture dish	Greiner Bio-One	664160
96 well round bottom plate	Thermo Scientific	163320
Alternative 96 well round bottom plate	Greiner Bio-One	650180
FP 30 syringe filter 0.45 μm	Whatman	10462656
BD LSRFortessa™ Cell Analyzer	BD Biosciences	N/A
BioTek 800 TS microplate reader	Agilent	N/A

Materials and equipment

BT474 and MCF7 culture medium

Reagent	Final concentration	Amount
DMEM High Glucose (4.5 g/l)	N/A	222.5 mL
Ham’s F-10	N/A	222.5 mL
FBS	10%	50 mL
Penicillin (10 000 Units/mL) -Streptomycin (10 mg/mL) Solution	Penicillin (100 Units/mL) Streptomycin (0.1 mg/mL)	5 mL
Total	N/A	500 mL

Store at 4°C for up to 6 months.

MDA-MB-361 culture medium

Reagent	Final concentration	Amount
DMEM High Glucose (4.5 g/l)	N/A	445 mL
FBS	10%	50 mL
Penicillin (10 000 Units/mL) -Streptomycin (10 mg/mL) Solution	Penicillin (100 Units/mL) Streptomycin (0.1 mg/mL)	5 mL
Total	N/A	500 mL

Store at 4°C for up to 6 months.

KHYG-1 culture medium

Reagent	Final concentration	Amount
RPMI 1640	N/A	435 mL
Heat inactivated FBS	10%	50 mL
Sodium pyruvate (100 mM)	1 mM	5 mL
L-glutamine (200 mM)	2 mM	5 mL
IL-2 (10 μg/mL)	4.6 ng/mL	230 μL
Penicillin (10 000 Units/mL) -Streptomycin (10 mg/mL) Solution	Penicillin (100 Units/mL) Streptomycin (0.1 mg/mL)	5 mL
Total	N/A	500 mL

Store at 4°C for up to 6 months.

Phoenix AMPHO culture medium

Reagent	Final concentration	Amount
DMEM high glucose	N/A	430 mL
FBS	10%	50 mL
Sodium pyruvate (100 mM)	1 mM	5 mL
L-glutamine (200 mM)	2 mM	5 mL
Non-essential amino acids (100X)	1X	5 mL
Penicillin (10 000 Units/mL) -Streptomycin (10 mg/mL) Solution	Penicillin (100 Units/mL) Streptomycin (0.1 mg/mL)	5 mL
Total	N/A	500 mL

Store at 4°C for up to 6 months.

ADCC assay medium

Reagent	Final concentration	Amount
RPMI 1640 media	N/A	450 mL
Heat inactivated FBS	5%	25 mL
Sodium pyruvate (100 mM)	1 mM	5 mL
L-glutamine (200 mM)	2 mM	5 mL
IL-2 (10 μg/mL)	4.6 ng/mL	230 μL
Penicillin (10 000 Units/mL) -Streptomycin (10 mg/mL) Solution	Penicillin (100 Units/mL) Streptomycin (0.1 mg/mL)	5 mL
Total	N/A	500 mL

Store at 4°C for up to 6 months.

Step-by-step method details

Generation of KHYG-1 cell line stably expressing FCγRIIIa (CD16)

Timing: 2 weeks

Immortalized natural killer cell lines, including the KHYG-1 cell line, either do not express Fc receptor FcyRIIIa (CD16) or rapidly lose its expression in culture.^4,5 CD16 expression is essential for recognition of therapeutic antibodies and killing of cancer cells through antibody-dependent cellular cytotoxicity (ADCC). Two variants of this receptor have been described, CD16 variant V158 shows increased antibody binding and increased cytotoxicity versus the F158 variant.^6,7 This step describes the generation of a natural killer cell line stably expressing CD16 (V158) that induces potent and specific ADCC. Starting with the generation of retroviruses with a CD16-V158 variant expression construct (Figure 2A), these retroviruses are used to transduce KHYG-1 natural killer cells and stably insert the CD16-V158 construct into the genomic DNA. Cell line KHYG-1 (DSMZ ACC 725) was established and described in 1997¹ and obtained from Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany.

Note: Use freshly thawed phoenix AMPHO cells at around 80% confluence. Seed phoenix AMPHO cells in antibiotic free medium before transfection.

Day 1.

• 1.
  For the reverse transfection prepare DNA lipofectamine2000 complexes.

  • a.
    Add 12 μg of pMSCV_IRES_GFP_CD16a vector to 1.5 mL optimem in a 15 mL tube. Mix by tapping gently.
  • b.
    Add 36 μL lipofectamine 2000 to 1.5 mL optimem in a 15 mL tube. Mix by tapping gently and incubate at 20°C–25°C for 5 min.
  • c.
    Add the diluted lipofectamine 2000 to the diluted DNA. Mix by tapping gently.
  • d.
    Incubate for 20 min at 20°C–25°C. Gently mix after 10 min.
• 2.
  During DNA lipofectamine incubation prepare the phoenix AMPHO cells.

  • a.
    Trypsinize and collect the phoenix AMPHO cells in a 15 mL tube.
  • b.
    Centrifuge at 210 × g for 5 min.
  • c.
    Count the cells and resuspend cells to reach 1.2∗10⁶ cells/mL in antibiotic-free medium.
• 3.Add 5 mL antibiotic-free medium to a 10 cm cell culture plate pre-coated with 0.1% gelatin.
• 4.Add DNA lipofectamine complexes in a drop wise fashion. Gently distribute the complexes in the plate with a cross-like movement.
• 5.Add 5 mL of cell suspension (6∗10⁶ cells) to the plate. Gently distribute the cells in the plate with a cross-like movement.
• 6.Place in a 37°C 5% CO₂ incubator.

Note: From now on retrovirus particles will be present. Work with the appropriate biosafety precautions.

Day 2.

• 7.
  Replace medium 24 h after transfection.

  • a.
    Carefully remove and discard the medium.
  • b.
    Gently add 6 mL KHYG-1 culture medium without IL-2 or antibiotics.
  • c.
    Incubate overnight in a 32°C 5% CO₂ incubator.

Note: As the vector contains GFP, fluorescent microscopy can be used to assess transfection efficiency.

CRITICAL: Virus collection medium should be similar to the medium used for culturing the KHYG-1 cells, as this medium will be used for transduction of the KHYG-1 cells.

CRITICAL: Use 6 ml medium for viral collection as this is the minimum to cover a 10 cm dish and will result in a high viral titer.

CRITICAL: Viruses are more stable at 32°C. Producing the virus at 37°C will reduce the transduction efficiency.⁸

Day 3.

• 8.
  Collect 48 h viral medium.

  • a.
    Collect all medium (around 6 mL) from the phoenix cells and place into a 50 mL tube.

    Optional: Fresh medium can be added to the phoenix cells to collect 72 h viral medium.

  • b.
    Centrifuge the viral medium at 400 × g for 5 min to pellet any cell debris.
  • c.
    Filter the viral medium using a 0.45 μm filter.
  • d.
    Add 2.8 μL IL-2 (10 μg/mL) to 6 mL viral medium for a final concentration of 4.6 ng/mL.

Note: Viral medium is best used directly but can be stored at 4°C for up to 1 week.

• 9.
  Transduction of KHYG-1 cells.

  • a.
    Equilibrate the viral medium to 20°C–25°C.
  • b.
    Transfer 2 mL viral medium to a new 15 mL tube and add 1.6 μL of polybrene (stock 10 mg/mL) for a final concentration of 8 μg/mL.

    Note: Working polybrene concentration varies for different cell lines. In our experience 8 μg/ml works well for KHYG-1 cells.

  • c.
    Count KHYG-1 cells and prepare two 15 mL tubes with 2∗10⁶ cells. Centrifuge at 210 × g for 5 min.

    Note: Use fresh KHYG-1 cells that have recovered from thawing (this takes about 2 weeks) with at least 95% cell viability. Seed cells at 0.4∗10⁶ cells/ml in antibiotic free medium the day before transduction.

    Note: Cell density at transduction is 0.5∗10⁶ cells/ml.

  • d.
    Remove supernatant and resuspend cells from one tube in 2 mL viral medium with polybrene and cells from the other tube in 2 mL growth medium, as a negative control.
  • e.
    Seed 2∗10⁶ cells in the middle wells of a 6-well plate.
  • f.
    Tape the plate to prevent spilling of the viruses.
  • g.
    Centrifuge the plate at 560 × g for 45 min with low acceleration (=1/9) and deceleration (=1/9). Use swinging buckets.
  • h.
    Remove the tape and place the plate in a 32°C 5% CO2 incubator for 16–24 h.

CRITICAL: IL-2 in the medium is essential for KHYG-1 maintenance and efficient transduction.

Note: Centrifuging the cells with viral medium greatly improves transduction efficiency.

Day 4.

• 10.Collect control and transduced cells in a 15 mL tube and centrifuge at 210 × g for 5 min.
• 11.Remove supernatant, count the cells and seed in antibiotic free KHYG-1 medium to a cell density of 0.5∗10⁶ cells/mL in a 6-well plate.
• 12.Place the cells back in the 32°C 5% CO₂ incubator.

Note: Use bleach to inactivate the viral particles in the discarded medium.

Day 5.

• 13.Collect control and transduced cells in a 15 mL tube and centrifuge at 210 × g for 5 min.
• 14.Remove supernatant and seed the transduced cells in 10 mL KHYG-1 medium with antibiotics into a 10 cm dish.
• 15.Place the cells in a 37°C 5% CO₂ incubator for 72 h to recover.

Day 8.

• 16.
  Start antibiotic selection of the transduced and control cells.

  • a.
    Count transduced and control KHYG-1 cells and seed at a density of 0.3∗10⁶ cells/mL in culture medium supplemented with 1 μg/mL puromycin.
• 17.Refresh medium with puromycin every 2–3 days. Monitor cell death of the transduced and control cells by resuspending the cells and counting them using trypan blue.

Note: Culture conditions can impact sensitivity to puromycin. It is recommended to do a puromycin titration in advance to select the optimal concentration for killing of the wild type cells. For example, in a 12-well plate seed 1 ml of 0.3∗10⁶ cells/ml cell suspension in 7 wells. To the wells add 0, 5, 10, 15, 20, 25 or 30 μl 10 mg/ml puromycin for a final concentration of 0, 0.5, 1, 1.5, 2, 2.5 or 3 μg/ml. Replace medium after 2 days. After 4 days assess cell death. The optimal puromycin concentration for selection is the lowest concentration at which all wild type cells are dead.

Day 15.

• 18.After 7 days all control cells should be dead. Expand the transduced cells in KHYG-1 culture medium until freezing.
• 19.Freeze 2∗10⁶ cells/mL in 20% FBS and 10% DMSO KHYG-1 culture medium as described in step 5 of culturing of KHYG-1 (CD16+) cells.

CD16 expression analysis

Timing: 2 h

This step describes how to monitor the expression of FcyRIIIa (CD16) on the cell surface of KHYG-1 cells after transduction and selection with puromycin. The construct used for transduction also contains the EGFP gene connected to the gene for selection (pac) with an IRES element. GFP fluorescence can be used as a reporter of the integration of the construct in the genomic DNA of the KHYG-1 cells (Figure 2A).

• 20.Collect wild type KHYG-1 and transduced KHYG-1 cells in a 15 mL tube.
• 21.Centrifuge at 250 × g for 2 min at 20°C–25°C.
• 22.Remove supernatant, resuspend cells in 2 mL PBS and count.
• 23.Move 6∗10⁴ cells into two 1.5 mL tubes per cell line.
• 24.Centrifuge at 250 × g for 2 min at 20°C–25°C.
• 25.
  Prepare antibody dilutions (in the dark, on ice).

  • a.
    Add 2 μL IgG-PE or CD16-PE in 50 μL PBS 1% BSA per sample.
• 26.Resuspend cells in 50 μL IgG-PE or CD16-PE dilution.
• 27.Incubate for 45 min at 4°C in the dark while rotating.
• 28.Wash 3 times with 200 μL PBS 1% BSA.
• 29.Resuspend cells in 0.5 mL PBS 1% BSA and transfer cells to a flow cytometry tube.
• 30.Analyze GFP and PE signal by flow cytometry using the LSRFortessa.

Alternative: The same procedure can be performed in a 96-well plate when handling a lot of samples.

Co-culture setup

Timing: 2 h

This step describes how to prepare and plate effector and target cells and antibody solutions for the ADCC assay (Figure 3 and 4). Initially, a serial dilution of trastuzumab can be performed to determine optimal antibody concentration (Figure 4A). We have determined a concentration 1 μg/mL of trastuzumab as the lowest concentration to obtain the highest cytotoxicity from KHYG-1 CD16+ cells in co-culture assays using 1∗10⁴ BT474 target cells at a 5:1 effector:target ratio (Figure 5A). This concentration is used for comparing different target and effector cell lines and effector:target ratios (Figure 4B).

Figure 3.

Schematics for co-culture conditions

Conditions required in each plate (preferably as triplicates in 3 wells) are: medium only (wells A–C in column 1) and medium with lysis buffer (wells D–F in column 1) to measure the medium background signal, target cells with antibody to measure target spontaneous release (column 3), lysed target cells to measure the target maximum release (column 4), natural killer cells with antibody to measure effector spontaneous release (column 2), co-culture of effector and target cells without antibody for detecting nonspecific cytotoxicity (column 5) and co-culture of effector and target cells with antibody for antibody-dependent cellular cytotoxicity (ADCC) (column 6).

Figure 4.

Schematics for co-culture plate design using different antibody concentrations or different target:effector ratios

(A) Plate design to assess antibody serial dilutions with a fixed target:effector ratio.

(B) Plate design to assess different target:effector ratios using a fixed antibody concentration. In both cases the steps are: 1) Plating of target cell lines (light and dark brown refer to 2 different target cell lines). Medium (pink) is added to wells without target cells. 2) Addition of only medium (pink) or antibody (yellow). 3) Seeding of KHYG-1 CD16+ cells (purple) at fixed ratio for antibody titrations experiments (A) or at different effector:target ratios (B). Lysis buffer is added to wells D–F in column 1 and wells A–F in column 4 (A) or wells A–F in column 6 (B) 45 min before the end of the co-culture incubation.

Figure 5.

Co-culture conditions influencing LDH release and cytotoxicity

(A) Normalized cellular cytotoxicity of KHYG-1 CD16+ cells on BT474 target cells in the presence of different antibody concentrations (0.001–2 μg/mL trastuzumab) at an effector:target ratio of 5:1 (n = 2). Error bars represent standard error of the mean (SEM).

(B) IL-2 addition to co-culture assay medium is not required for cellular cytotoxicity of KHYG-1 CD16+ cells against BT474 target cells.

(C) Background LDH release of natural killer cells in medium with different concentrations of FBS after subtraction of the only medium background signal (n = 3).

(D) Presence of trastuzumab during 24 h culture of target or effector cells does not alter their LDH release. LDH release is normalized to cells cultured without antibody (n = 3). Error bars represent standard error of the mean (SEM).

(E) LDH release of 1∗10³ to 4∗10⁴ BT474 (blue), MCF7 (green) and MDAMB361 (red) cells with or without lysis buffer.

(F and G) LDH release of KHYG-1 CD16+ and BT474 alone or in co-culture after 6 h (F) or 24 h (G) incubation.

(H) For the BT474 cell line, but not for the MDA-MB-361 cell line, antibody-dependent KHYG-1 CD16+ mediated cell killing is enhanced by increasing assay time from 6 h to 24 h.

We recommend using a range of 5∗10⁴ to 2∗10⁵ target cells per well. Decreasing the cell number reduces the detection window between the target cell background LDH release and the target cell maximum (Figure 5E). This will compromise the ability to detect cytotoxicity changes. The effector:target ratio is limited by the concentration of KHYG-1 cells as exceeding 1∗10⁶ cells/mL results in reduced proliferation and loss of cytotoxicity.

Note: To minimize pipetting errors and produce more reliable results, use a multichannel pipet for transferring cells, antibody or medium to the 96-well plate.

Note: Always use triplicates for each co-culture condition.

Optional: IL-2 preactivation enhances ADCC,⁹ but is not absolutely required during the assay (Figure 5B).

• 31.Calculate the number of cells needed for the experiment taking into account that 1∗10⁴ target cells will be seeded per well in a 96-well plate.
• 32.
  Prepare target cells suspension.

  • a.
    Harvest target cells (BT474, MCF7 or MDA-MB-361).
  • b.
    Centrifuge at 210 × g for 5 min.
  • c.
    Resuspend cells in ADCC assay medium and count.
  • d.
    Prepare a cell suspension of target cells of 2∗10⁵ cells/mL.
• 33.Plate 50 μL of target cell suspension in the appropriate wells of a round bottom 96-well plate to get 1∗10⁴ cells per well (Figure 4).

CRITICAL: Use a round bottom 96-well plate to ensure interaction between the effector and target cells. A flat bottom plate will yield no to little cytotoxicity in this setup.

• 34.Add 50 μL ADCC assay medium to wells without target cells.
• 35.
  Prepare antibody dilutions.

  • a.
    Prepare a 10× dilution of trastuzumab for a stock concentration of 2.1 mg/mL.
  • b.
    Prepare 2× concentrated working stocks of trastuzumab, because the trastuzumab will be diluted 1:2 when added to the wells containing 50 μL medium.

    Note: Trastuzumab is also added to control wells with only target cells or effector cells. This does not alter the viability of these cells as measured by LDH release (Figure 5D).

    • i.
      For Figure 4A - antibody serial dilutions: First prepare a 4 μg/mL working stock by adding 1.4 μL trastuzumab (2.1 mg/mL) to 750 μL ADCC assay medium. To 7 wells of a 96-well plate add 175, 154, 107, 225, 116 and 145 μL ADCC assay medium. Add 320 μL of 4 μg/mL working stock to an 8^th well and serial dilute into the next well by transferring 145, 116, 56, 107, 39 and 18 μL sequentially following the scheme in Figure 6.
    • ii.
      For Figure 4B - fixed antibody concentration: Prepare a 2 μg/mL working stock by adding 2.4 μL trastuzumab (2.1 mg/mL) to 2.5 mL ADCC assay medium.
• 36.Add 50 μL of the antibody working stocks to the appropriate wells (Figure 4).
• 37.Add 50 μL ADCC assay medium to wells without antibody.
• 38.Place the plate in a 37°C 5% CO₂ incubator for 30 min.
• 39.
  In the meantime prepare effector cell suspensions.

  • a.
    Collect the effector cells in a 15 mL tube.
  • b.
    Centrifuge at 210 × g for 5 min.
  • c.
    Resuspend cells in ADCC assay medium and count.
  • d.
    Prepare effector cell suspensions.

    • i.
      For a 1:1 effector:target ratio, prepare a cell suspension of 1∗10⁵ cells/mL. For 1 plate with 2 cell lines resuspend 3∗10⁵ cells in 3 mL assay medium and plate 1∗10⁴ effector cells (100 μL) per well.
    • ii.
      For a 5:1 effector:target ratio, prepare a cell suspension of 5∗10⁵ cells/mL. 3∗10⁵ cells in 3 mL. For 1 plate with 2 cell lines resuspend 1.5∗10⁶ cells in 3 mL assay medium and plate 5∗10⁴ effector cells (100 μL) per well.
    • iii.
      For a 10:1 effector:target ratio, prepare a cell suspension of 1∗10⁶ cells/mL. For 1 plate with 2 cell lines resuspend 3∗10⁶ cells in 3 mL assay medium and plate 1∗10⁵ effector cells per well.
• 40.Add 100 μL of effector cell suspension to the appropriate wells (Figure 4).
• 41.Add 100 μL of assay medium to the wells without effector cells.
• 42.Centrifuge the plate at 250 × g for 4 min.

CRITICAL: Do not centrifuge faster than 250 × g as this can affect effector cell viability.

Note: This step is performed to ensure contact of the effector cells and target cells in the bottom of the plate.

• 43.Place the plate in a 37°C 5% CO₂ incubator for 6 or 24 h.
• 44.45 min before the end of the incubation time, add 20 μL lysis buffer to the maximum release wells with target cells and 3 wells with only medium (Figure 3). For plate setup in Figure 4A, add lysis buffer to wells D-F1 and A-F4. For plate setup in Figure 4B, add lysis buffer to wells D-F1 and A-F6.

Optional: 6 h is enough time to see antibody-dependent cellular cytotoxicity. For some, but not all cell lines, cytotoxicity can be enhanced by increasing the assay time to 24 h (Figures 5F, 5G, and 5H).

Figure 6.

Serial dilution of trastuzumab in ADCC assay medium

Cytotoxicity measurement and analysis

Timing: 1 h

Cell lysis by ADCC results in the release of the cytosolic enzyme lactate dehydrogenase (LDH) into the medium. This step describes the measurement of LDH release after the ADCC assay adapted from the Promega CytoTox 96 assay, and how to normalize the data and calculate the percentage of cytotoxicity.

• 45.Thaw cytotox96 reagent and keep on ice in the dark.
• 46.Centrifuge the ADCC plate at 250 × g for 4 min.
• 47.Transfer 50 μL supernatant to a fresh 96-well plate.
• 48.Add 50 μL cytotox96 reagent to each well.
• 49.Incubate 30 min at room temperature in the dark.
• 50.Add 50 μL stop solution (1M acetic acid).
• 51.Pop large bubbles using a syringe needle.
• 52.Measure absorbance at 490 nm within 1 h using a plate reader.

Note: The LDH release assay results in a minimum of 7 experimental values: medium background, medium + lysis background, target spontaneous release, effector spontaneous release, target maximum release, nonspecific cytotoxicity (target and effector cells without antibody) and antibody dependent cellular cytotoxicity (target and effector cells with antibody) (Figure 3).

• 53.
  Background subtraction.

  • a.
    Calculate the average signal of the medium only wells (Figures 3 and 4, wells A-C1) and the medium + lysis buffer wells (Figures 3 and 4, wells D-F1).
  • b.
    Subtract the average medium background (Figures 3 and 4, wells A-C1) from all wells, except the maximum LDH release wells (Figures 3 and 4A, wells A-F4; Figure 4B, wells A-F6). For the maximum LDH release wells, subtract the average medium + lysis buffer background (Figures 3 and 4, wells D-F1).

Note: The background signal of the medium and spontaneous release is partially influenced by the percentage or composition of the FBS. However, no difference in spontaneous LDH release by KHYG-1 CD16+ cells is observed under different FBS conditions after background subtraction (Figure 5C).

• 54.
  Calculate cytotoxicity.

  • a.
    Calculate the average of all triplicates required in the formula below.

    • i.
      For the ‘experimental lysis’ calculate the average of the nonspecific cytotoxicity wells (Figure 3, wells A-C5 and D-F5) and the ADCC wells (Figure 3, wells A-C6 and D-F6).
    • ii.
      For the ‘effector spontaneous’ calculate the average of the ‘effector cells spontaneous LDH release’ wells (Figure 3, wells A-F2).
    • iii.
      For the ‘target spontaneous’ calculate the average of the ‘target cells spontaneous LDH release’ wells (Figure 3, wells A-C3 and D-F3).
    • iv.
      For the ‘target maximum’ calculate the average of the ‘max LDH release’ wells (Figure 3, wells A-C4 and D-F4).
  • b.
    Calculate the cytotoxicity of the co-culture wells with or without antibody using the following formula:

$$\%cytotoxicity=\frac{Experimentallysis-Effectorspontaneous-Targetspontaneous}{Targetmaximum-Targetsponaneous}\ast 100$$

Note: The percentage of cytotoxicity indicates what proportion of the cells were killed by the natural killer cells during the co-culture experiment. When all target cells are dead the cytotoxicity is 100%.

Note: Spontaneous LDH release of the natural killer cells can be high. However, this does not impact the calculated percentage of (nonspecific or antibody-dependent) cytotoxicity as the LDH release values in the co-culture wells are adjusted for spontaneous release of the target and natural killer cells.

Optional: For calculating cytotoxicity to determine optimal antibody concentration (Figures 4A and 5A), normalize the co-culture data from 0–100% by using the following formula:

$$\%normalizedcytotoxicity=\frac{Experimentallysis-Noantibodylysis}{Highestantibodyconcentrationlysis-Noantibodylysis}\ast 100$$

Expected outcomes

Immortalized KHYG-1 cells have lost expression of the FcγRIIIa (CD16),⁴ which is required to mediate ADCC. Transduction of KHYG-1 to generate a stable CD16-overexpressing cell line is expected to yield around 95% GFP and CD16 positive cells after 1 week of selection. More importantly, CD16 expression is stable for at least 3 months (Figure 2B).

The ADCC assay measures LDH release in the medium as a readout of cell lysis and therefore cell killing of target cells by effector cells. After background subtraction, a clear difference is seen between spontaneous LDH release of the target cells and the maximum LDH release of the target cells (Figure 7A). For most target cells the spontaneous LDH release is almost negligible. This difference provides a suitable window to measure the cytotoxicity that is exerted from the natural killer cells on the target cells. On the other hand, KHYG-1 CD16+ cells have more spontaneous LDH release than the target cells. Depending on the number of effector cells used in the assay the background increases and this value needs to be subtracted to calculate the percentage of cytotoxicity.

Figure 7.

Expected outcomes of the ADCC assay

(A) LDH release of KHYG-1 CD16+ and BT474 alone or in co-culture.

(B) Cytotoxicity of KHYG-1 wild type or KHYG-1 CD16+ cells on BT474 cells.

(C) Cytotoxicity of KHYG-1 CD16+ cells against HER2-overepressing BT474 cells (n = 3) and HER2-low MCF7 cells (n = 4) at an effector:target ratio of 5:1. ns is defined as p>0.05.

(D) Cytotoxicity of KHYG-1 CD16+ cells against HER2-low MCF7 cells at an effector:target ratio of 1:1 (n = 1), 5:1 (n = 4) and 10:1 (n = 2). ns is defined as p>0.05.

(E) LDH release of KHYG-1 CD16+ or PBMCs at different effector:target ratios cultured alone or with BT474 cells as target cells.

(F) Cytotoxicity of KHYG-1 CD16+ cells and PBMCs against BT474 cells (n = 3). Error bars represent standard error of the mean (SEM). ns is defined as p>0.05.

Co-culture of KHYG-1 CD16+ cells with target cells in the absence of antibodies results in a slight increase of LDH release due to the nonspecific cytotoxicity of KHYG-1 CD16+ cells on the target cells. With the addition of an antibody the natural killer cells are activated and exert potent and specific antibody-dependent cellular cytotoxicity towards the cancer cells (Figure 7A).

The antibody we use in this protocol is trastuzumab, which is the first line treatment for HER2-positive cancer patients. This HER2-targeting agent binds HER2-overexpressing cancer cells and the Fc receptor in natural killer cells enhancing the antitumor immune response towards HER2-overexpressing cancer cells.^10,11,12 Indeed using the KHYG-1 CD16+ natural killer cell line we achieve 100% cell killing of the HER2 overexpressing BT474 cell line at 5:1 effector:target ratio with 1 μg/mL trastuzumab, while nonspecific cytotoxicity is nearly 0% (Figure 7B). As expected, against HER-low breast cancer cell line MCF7 the addition of trastuzumab to the co-culture assays yields low antibody-dependent cytotoxicity using different effector:target ratios (Figures 7C and 7D). Thus, the KHYG-1 CD16+ cell line is a suitable tool to measure specific ADCC against HER2-positive breast cancer cells.

PBMCs are often used for ADCC assays. The KHYG-1 CD16+ cell line can be used as an off the shelf standardized scalable replacement for the use of PBMCs, which are laborious to produce and have high donor variability. Of the PBMCs only a subset of natural killer cells express the CD16 receptor, can recognize trastuzumab using this receptor and are capable of ADCC. The fraction of natural killer cells in the PBMCs varies between 5 to 20%.³ An effector:target ratio of 20:1 for PBMCs is therefore comparable to a 1:1 to 4:1 ratio of natural killer cells. Importantly, when comparing between these ratios the cytotoxicity of the KHYG-1 CD16+ cell line is similar to cytotoxicity exerted by PBMCs (Figures 7E nd 7F).

Limitations

Our protocol has successfully produced a KHYG-1 CD16+ cell line, available upon request, capable of potent and specific antibody-dependent cellular cytotoxicity comparable to human PBMCs. This protocol can be used to generate other natural killer cell lines stably expressing CD16, such as the NKL cell line, albeit further optimization might be required.

In this protocol cytotoxicity is measured using the relatively expensive Promega CytoTox 96 assay. A cheaper alternative is the use of a luciferase-based cytotoxicity assay. However, this assay requires the generation of target cells that stably express luciferase.^13,14

Cytotoxicity is highly dependent on good laboratory practice. For the most accurate result, precise pipetting, counting of the cells, proper culture of the KHYG-1 CD16+ cell line, and controls in each experiment are essential.

Troubleshooting

Problem 1

Inefficient transduction of natural killer cells or no surviving cells after selection. (Production of a stable CD16 overexpressing KHYG-1 cell line)

Potential solution

• •Use phoenix amphotropic cell line to produce amphotropic retroviruses capable of transducing human cell lines.
• •Check the transfection efficiency of the phoenix cells using fluorescence microscopy if the cDNA construct contains a fluorescent reporter cassette.
• •If many phoenix cells are detached from the plate, this will negatively impact virus production. Use gelatin coated plates to promote cell attachment. Very gently remove and add medium and use pre-warmed medium.
• •Transfect enough phoenix cells. Too low confluence of phoenix cells will result in more cell death after transfection and consequently, lower virus production. The plate should be around 80–90% confluent 24 h after transfection. During virus production the cells are placed at 32°C which will limit cell proliferation.
• •Concentrating the virus by ultracentrifugation can increase viral titer. Although correctly transfected phoenix cells should produce enough viruses for transduction without concentrating.
• •Use plasmid DNA of high quality and purity.

Problem 2

KHYG-1 (CD16+) cells proliferate slowly or show high cell death (over 10%). (Culturing of KHYG-1 (CD16+) cells)

Potential solution

• •
  KHYG-1 (CD16+) cells have grown overconfluent.

  • ○
    Re-seed the cells and allow KHYG-1 CD16+ to recover for 2 weeks and test cytotoxicity on a previously tested target cell line.
  • ○
    Thaw a new vial of KHYG-1 CD16+ cells.
• •
  Medium composition is incorrect or it has been improperly stored. Especially, lack of IL-2 in the medium will cause stress on the KHYG-1 cells and complete absence of IL-2 will lead to cell death within 4 days.

  • ○
    Prepare fresh medium. Use aliquots of IL-2 stored at −20°C for preparation of the medium.

Problem 3

Negative or very low cytotoxicity (Co-culture setup).

Potential solution

• •
  KHYG-1 CD16+ cells have been cultured in suboptimal conditions. This can result in a higher LDH spontaneous release of the natural killer cell alone than in co-culture with target cells. Additionally, cell killing of the target cells by the natural killer cells will be less efficient.

  • ○
    Allow cells to recover. Make sure cell density does not exceed 1∗10⁶ cells/mL. Seed cells in fresh medium every 2–3 days.
  • ○
    Thaw a new vial of KHYG-1 CD16+ cells.
• •Incomplete cell lysis in the maximum release wells. Check the cell lysis using a microscope and add more lysis buffer if lysis is incomplete.
• •Low expression of the antigen in the target cells. Check the expression of the antigen that is targeted by the antibody used in the assay. In this study the antigen is HER2.
• •Not enough antibody or natural killer cells were added. Test different antibody and natural killer concentrations.
• •The assay time was too short. Increase co-culture incubation time from 6 h to overnight or 24 h to allow for more cytotoxicity.

Problem 4

Clear outliers are present after LDH measurement. Single wells differ largely from their replicates and/or red precipitate is visible in the wells. (LDH release measurement)

Potential solution

• •Precipitate is present in the cytotox96 reagent or buffer. Before using the reagent or buffer, centrifuge at 300 × g for 5 min and transfer reagent or buffer to a new tube.
• •
  Cells were accidentally transferred during the transfer of medium from the co-culture plate to the LDH measurement plate.

  • ○
    Centrifuge the co-culture plate before transferring medium.
  • ○
    Pipet carefully while transferring the medium. Do not resuspend the medium.
• •Large bubbles are present in the wells. Pop large bubbles with a needle.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Ana Ruiz-Saenz (a.ruizsaenz@erasmusmc.nl).

Materials availability

KHYG-1 CD16+ cell line and CD16-containing plasmid described in this study will be made available from the lead contact for academic/noncommercial research purposes on request under appropriate materials transfer agreement.

Plasmids used in this study have been deposited to Addgene: pMSCV_IRES_GFP_CD16a #196189.

Data and code availability

This study did not generate unique datasets or code.