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:

  1. Don't disturb the 721.221.mIL21 cell clusters, and harvest the 721.221.mIL21 cells by serological pipet.
  2. Resuspend the 721.221.mIL21 cells from the bottom and transfer to 50ml canonical tube.
  3. 400g, 5mins, RT. Discard the supernatant.
  4. 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)
  5. 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)
  6. Count the cells and aliquot the 721.221.mIL21 cells into 10 million per vial in the frozen media.
  7. 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.