721.221-mIL21 Feeder Cell Irradiation Protocol
Hsiang-chi Tseng and Dongfang Liu (Rutgers-NJMS)
11-03-2021
Reagents and Instrument:
Item |
Working Concentration |
Manufacturer |
Catalog # |
Comment |
location |
Sterile PBS |
|
Corning |
21-040-CM |
|
|
RPMI-1640 |
|
Corning |
15-040-CV |
|
|
Glutamax |
1% |
Gibco |
35050061 |
|
|
FBS |
10% |
Corning |
35-010-CV |
|
|
Frozen Media |
95% FBS + 5% DMSO |
DMSO
Sigma D2650 |
|
|
Irradiator |
JL Shepherd & Associates 137Cs irradiator, Model Mark I-68 |
CC-I1105 |
Note: Complete media = RPMI-1640 with 10% FBS and 1% of Glutamax
Experimental Purpose: irradiate 221.mIL21 feeder cells to expand NK and CAR-NK cells (key step for successful NK and CAR-NK expansion). For more details, please check our publication (Yang, Y. et al., 2020)1.
Step-by-Step Procedures:
- Don't disturb the 721.221.mIL21 cell clusters, and harvest the 721.221.mIL21 cells by serological pipet.
- Resuspend the 721.221.mIL21 cells from the bottom and transfer to 50ml canonical tube.
- 400g, 5mins, RT. Discard the supernatant.
- Resuspend in PBS, the final concentration is less than 4 X 10^6/ml. (In my case, I collected from ten T-75 flasks and resuspended all the cells in 50 ml PBS in one 50 ml canonical tube)
- Put the 50 ml canonical tube on ice and ready for 100GY irradiation. (The irradiator we are using is listed above; there will be a carousel next to the irradiator that we can put the 50ml canonical on and rotate inside the chamber during irradiation)
- Count the cells and aliquot the 721.221.mIL21 cells into 10 million per vial in the frozen media.
- Ready to use for NK and CAR-NK expansion.
References:
1. Yang Y, Badeti S, Tseng HC, Ma MT, Liu T, Jiang JG, et al. Superior Expansion and Cytotoxicity of Human Primary NK and CAR-NK Cells from Various Sources via Enriched Metabolic Pathways. Mol Ther Methods Clin Dev 2020; 18:428-45.
