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Document # CD-OP006 08/22© 2020 Lonza Walkersville, Inc.

Natural Killer (NK) Cell Expansion/Activation andCytotoxicity Assay
Instructions for use
Safety statements

THESE PRODUCTS ARE FOR RESEARCH USE ONLY. Not
approved for human or veterinary use, for application to humans
or animals, or for use in clinical or in vitro procedures.
WARNING: LONZA PRIMARY CELLS CONTAIN HUMAN
SOURCE MATERIAL; TREAT AS POTENTIALLY INFECTIOUS.
Each donor is tested and found non-reactive by an FDA-approved
method for the presence of HIV-I, hepatitis B virus and hepatitis Cvirus. Where donor testing is not possible, cell products are tested
for the presence of viral nucleic acid from HIV-1, hepatitis B virus,
and hepatitis C virus. Testing cannot offer complete assurance
that HIV-1, hepatitis B virus, and hepatitis C virus are absent. All
human-sourced products should be handled at the biologicalsafety level 2 to minimize exposure to potentially infectious
products, as recommended in the CDC-NIH manual, Biosafety in
Microbiological and Biomedical Laboratories, 5 th edition. If you
require further information, please contact your site safety officer
or Scientific Support.

Preparation of reagents for NK Cell assay

All work should be done in a laminar flow hood.
Decontaminate the external surfaces of all
supplement vials and the medium bottles withethanol or isopropanol.
1. IL-15 Working Solution
Make IL-15 working solution by adding 10 µL of100 µg/mL IL-15 working solution to 990 µL H20
for a working concentration of 1 µg/mL IL-15 (IL15 is used to trigger NK cell expansion).
2. IL-21 Working Solution
Make IL-21 working solution by adding 10 µL of
100 µg/mL IL-21 working solution to 990 µL H20
for a working concentration of 1 µg/mL IL-21 (IL21 is used to boost NK cell stimulatory effect).
3. CellTracker Green Dye Working Solution
NOTE: A llow dye to get to room temperature prior toreconstitution of lyophilized product.

Reconstitute lyophilized product in 200 µL
DMSO to make a stock concentration of 10mM.
Make a working solution by adding 0.5 µL of the
10mM stock to 999.5 µL X-VIVO® 15 Serumfree Hematopoietic Cell Medium for a working
concentration of 5 µM.

NK Cell Expansion and Activation Protocol
NOTE: All work is to be performed in a laminar flow hood.

1. Pipette 10 mL of pre-warmed X-VIVO® 15Serum-free Hematopoietic Cell Medium into 15
mL conical tube and place in laminar flow hood.
2. Thaw NK cells at 37oC for ~2 min until only asliver of ice remains and pipette cells intoX-VIVO® 15 Serum-free Hematopoietic CellMedium in 15 mL conical from step 1.
3. Wash cell vial with 1 mL X-VIVO® 15 Serumfree Hematopoietic Cell Medium from conicaland add back to conical.
4. Centrifuge cells at 300xg for 10 minutes at 4oC
5. Resuspend pellet in 1 mL X-VIVO® 15 Serumfree Hematopoietic Cell Medium and count using
Trypan Blue and a hemocytometer at a 1:50
dilution.
6. After counting, resuspend cells at 2 x 106
cells/mL in X-VIVO® 15 Serum-freeHematopoietic Cell Medium and plate cells withIL-15a. In 24-well plate: Add 1 mL of cellsuspension to each well, then add 10 µL
of IL-15 working solution
b. In 48-well plate: Add 500 µL of cellsuspension to each well, then add 5 µLof IL-15 working solution
7. Incubate cells at 37oC, 5% CO2 for 10 days withmedia change every 3-4 days. Follow thefollowing sub-steps to change media
a. Collect all cells and supernatant into a15 or 50 mL conical tube.
b. Centrifuge the cells at 300xg for 10minutes at room temperaturec. After centrifugation, aspirate thesupernatant and resuspend cell pellet inX-VIVO® 15 Serum-free Hematopoietic
Cell Medium at 1 mL/well or 500 µL/well

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for 24-well and 48-well culture plate,respectively.
d. Add cells back to the culture plate at 1mL/well or 500 µL/well for 24-well and48-well culture plate, respectively.
e. Add 10 µL/well or 5 µL/well of IL-15
working solution for 24-well and 48-wellculture plate, respectively.
f. Incubate cells at 37oC, 5% CO2
8. On day 10, follow through step 7 for media
change, then add 25 µL/well or 12.5 µL/well ofIL-21 working solution for 24-well and 48-wellculture plate, respectively.
9. Incubate cells until day 14, then harvest cells
and proceed with downstream assay(s) ofinterest.

•12. To prep NK cells for the assay, remove NK cells
and media from culture.
13. Centrifuge cells at 300xg for 10 minutes at room
temperature.
14. Resuspend cells in 1 mL pre-warmed X-VIVO®
15 Serum-free Hematopoietic Cell Medium andcount using Trypan Blue and a hemocytometer.
15. After counting, remove ~1 x 106 cells and add toa separate conical.
16. Fill cell suspension to 1 mL with X-VIVO® 15Serum-free Hematopoietic Cell Medium for a cellconcentration of ~1 x 106 cells/mL.

•NOTE: Immuno-phenotyping can be performed via FACS
on Day 0 and 14 targeting CD56, CD16, CD3, NKG2D,
NKp46 and NKp44 cell surface markers to assess
activation status of NK cells.

Cell Staining

17. After the appropriate preparation of both K-562
and NK cells, make 2 FACS tube for eachcondition of K562 cells and NK cells at thefollowing ratio:a. 1:1 (i.e. 100,000 K-562 cells : 100,000NK cells).
b. 1:0 (i.e. 100,000 K-562 cells only. This
will be used to account for spontaneous
K-562 cell death).

NK Cell Cytotoxicity Assay

Note: The NK cells used in this experiment need to be matured
and activated in culture for 14 days by following the NK Cell
Expansion and Activation protocols prior to initiating the NK Cell
Cytotoxicity Assay.
NOTE: K562 cells will be required for this assay. It isrecommended to plate and culture these cells at least 4-5 days
prior to initiating the NK Cytotoxicity Assay. Follow themanufacturer’s protocols for culturing K-562 cells.

•NK Cell Preparation

NOTE: A recommended minimum of 100,000 K-562
cells for FACS analysis should be taken intoconsideration.

K-562 Cell Preparation

1. Warm working solution of CellTracker dye to37oC.
2. While dye is warming, remove K562 cells andmedia from culture.
3. Centrifuge cells at 300xg for 10 minutes .
4. Resuspend cells in 1 ml X-VIVO® 15 Serumfree Hematopoietic Cell Medium and count cells
using Trypan Blue and a hemocytometer.
5. After counting, remove ~1 x 106 cells and add toa separate conical.
6. Fill cell suspension to 1 mL with X-VIVO® 15Serum-free Hematopoietic Cell Medium for a cellconcentration of ~1 x 106 cells/mL.
7. Add 1 µL working solution of CellTracker GreenDye to the cell conical.
8. Incubate at 37oC, 5% CO2 for 1 hour.
9. After incubation with CellTracker Green Dye(from step 7-8), add 2 mL of PBS to the conicaland centrifuge at 300xg for 5 minutes at room
temperature.
10. After centrifugation, aspirate or decant tubes toremove wash solution.
11. Resuspend cells in 1 mL pre-warmed X-VIVO®
15 Serum-free Hematopoietic Cell Medium.

18. Incubate cells for 3 hours at 37oC, 5% CO2.
19. After incubation, add 1 µL SYTOX dead cellstain to each tube.
20. Incubate tubes for 15 minutes at 4oC.
21. After incubation, add 1 mL of PBS to each tube
and centrifuge at 300xg for 5 minutes at 4oC
22. After centrifugation, decant tubes to remove
wash solution.
23. Resuspend cells in 1% PFA and analyze via
FACS.
24. Assess cells via the % Cell Death of K-562 cells
to determine cytotoxic effect of NK cells(increased K-562 % cell death, as compared tothe K-562-only control tubes, indicates high NK
cell cytotoxic effect).

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Ordering information
Catalog no.

Description

Size

2W-501NK - Human
Natural Killer
Cells,
Cryopreserved,
NegativeSelection≥5 million cells

04-418Q

X-VIVO® 15
Serum -freeHematopoietic
Cell Medium

1L, complete withL-Glutamine, gentamicin,
and phenol red, xenofree

12-702F

RPMI 1640
Medium

500 mL, with L-Glutamine,
phenol red

THESE PRODUCTS ARE FOR RESEARCH USE ONLY.
Not approved for human or veterinary use, for application tohumans or animals, or for use in clinical or in vitro procedures.

IL-15 (PeproTech P/N 200-15) mentioned is a product from
PeproTech.
IL-21 (PeproTech P/N 200-21) mentioned is a product fromPeproTech.
CellTracker™ Green CMFDA Dye (Thermo P/N C2925)
mentioned is a product from Thermo Fisher Scientific.
SYTOX™ Blue Nucleic Acid Stain (Thermo P/N S11348)
mentioned is a product from Thermo Fisher Scientific.
K-562 Cells (ATCC P/N CCL-243) mentioned is a product fromATCC.
All other trademarks herein are marks of Lonza Groupor its subsidiaries.

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