Detection of phospho-epitopes on human NK cells for cell signaling analysis

1-Thaw cryopreserved PBMCs

2-Distribute cells in sterile 96 well plates, 1 million per well.

3-Rest cells in complete RPMI medium (50ul) for 1 h at 37 C and 5% CO₂. 

4-Stimulate with 50ul of reconstituted cytokines or PBS (non-stimulated control) for 15 min (to test depending of panel) at 37 C and 5% CO₂ in the presence of LIVE/DEAD Yellow (Life Technologies).

5-Stop stimulation with 100ul of formaldehyde 4% (Polysciences), RT, 10 min.

6-Spin plate at 1600RPM, 3min, room temperature (RT), remove supernatant, and wash with 200ul FACS buffer // X2

7-Stain cells for extracellular markers in FACS buffer 50ul final volume. Include Fc receptor blocking (Miltenyi) in the antibody mix RT, 15min-30min.

8-Spin plate at 1600RPM, 3min, RT, remove supernatant, and wash with 200ul FACS buffer // X2

9-Resuspend pellet in 150ul 4degree methanol, place plate in -20 C, 20-30min.

10-Spin plate at 1600RPM, 3min, RT, remove supernatant, and wash with 200ul Perm/MQ buffer // X2

11-Stain for intracellular proteins (including phospho-epitopes) was performed in Perm/MQ, RT 45min- 2h (to test depending of panel). Include Fc receptor blocking (Miltenyi) in the antibody mix.

12-Spin plate at 1600RPM, 3min, RT, remove supernatant, and wash with 200ul Perm/MQ buffer // X2

13-Resuspend pellet in 200ul of FACS buffer. Acquire to flow.

Complete medium: Hyclone RPMI+L-Glutamine x 1+ 10% FCS

FACS Buffer: 1L PBS, 2mM EDTA+(4ml, ref : Ambion AM9260G),  2% FCS (20ml : ref : Sigma F7524)

*Fixation/permeabilisation buffer (Fix/perm buffer) :

dilution ¼ with eBioscience reagents

Fix/perm diluent : 00.5223.56 (100ml bottle)

Fix/perm concentrate : 00.5123.43 (30ml bottle)

**Permeabilisation buffer (perm/MQ buffer) :

dilution eBioscience reagent  1/10 with MQ water

Perm Buffer 10X 00.8333.56 (100ml bottle)

Category