ADCC LDH Protocol

Experiment: ADCC Target Cells by Primary NK cells (Whole Blood Donor Isolated PBMCs)

Materials:

Culture medium for NK cell activation: Myelocult H5100 (Stemcell 05150) with 1uM Hydrocortisone (STEMCELL 74142) and 1000 IU/mL IL-2 (R&D system BT002AFL01M).  (20 mL Myelocult + 1x Hydrocotrison .1 mL (1:200 from stock) + .8 uL IL-2 stock (1000 IU/uL)

Assay medium (pH7.4): AIM 5 Media with 1% FBS (no sodium pyruvate) (use low IgG FBS if doing LDH)

Frozen PBMC (HS31028, HS31021): 1.6e7 viable PBMC from 10 vials of frozen cells. 

NK cell isolation kit:  Stemcell Technologies

Procedures:

1.  Thaw 10 vials of frozen PBMC.  Spin down cells and resuspend in 20 mL of culture medium.  Count cells
2. Spin down cells and isolate NK cells using Stemcell NK cell Isolation Kit in accordance with manufacturer’s protocol. 
3. Resuspend NK cells in NK cell activation medium at 1e6 cells per mL and incubate overnight. 
4. Count cells.  Resuspend cells in assay medium at 2.78e5/mL.  Plate 90 uL Target Cells in U-bottom plate. 
5. Make serial dilution of antibodies (8-point, 10-fold starting from 100 ug/mL) (50uL)
6. Add 20 uL of antibody solution to the plate according to plate design. 
7. Wash NK cells once.  Count and resuspend NK cells in assay medium @ 2.78e5/mL.  Add 90 uL NK cells to the plate. 
8. Incubate culture plate at 37C 5% CO2 for  4 hours.  Take out 100 uL medium for LDH readouts in a flat bottom plate
9. LDH
   1. Reconstitute catalyst and incubate at RT for 10 mins
   1. Add catalyst to Dye 1
   1. In a flat bottom plate, add 100 uL dye to sample and incubate for 20 mins
   1. Add 50 uL stop solution and shake at 600 speed for 10 secs
   1. Immediately get readout on Spectramax
10. Perform graphpad analysis