Isolate CD4+ cells from spleen and/or lymph nodes using Stem Cell EasySep™ Mouse CD4+ T Cell Isolation Kit, Ref #19852.

Plate cells for polarization according to protocol.

Neutravidin resuspension:

Resuspended lyophilized 10 mg glass vial with 1 mL of ultrapure H2O (milliQ water) to make 10 ug/uL solution. Ensure that all neutravidin is dissolved, then dilute to 10 mL in 1X PBS. Make 1 mL aliquots and store at -20 deg C. Active aliquot can be kept at 4 deg C for weeks to months.

Anti-CD3 and anti-CD28 biotinylation:

Stock concentration of 2C11 (anti-CD3) from BioXcell: 2.8 mg/mL

Stock concentration of 37.51 (anti-CD28) from BioXcell: 2.0 mg/mL

1. Isolate naive T cells (see "Human T cell isolation protocol")
2. Activation and polarization - 4 days culture - day 0
   • Pre-coat plates with anti-hCD3 (1ug/ml; clone OKT3) and anti-hCD28 (2ug/ml; clone 15E8; UCSF core) in PBS. 1-2 hours at 37°C.
   • Culture T cells at 1x10⁶ cells/ml in standard culture medium
     • RPMI 1640 (invitrogen) + 10% FBS, 2mM L-glutamine, 100U/ml penicillin, 100g/ml streptomycin, 1mM sodium pyruvate.
   • Th1 cocktail:
     • rIL-12 (2.5ng/ml; R&D systems)

Lung digest to prepare cells for FACS analysis.

List of cytokines and antibodies used in mouse T cell activation/resting/differentiation

Includes vendor and catalogue numbers

1. T cells are grown as usual (plate-bound until 72 h, expanded in IL-2 until day 6)

2. Restimulation: Plate ~70 million cells in 2 10” Greiner cell culture plates. Leave ~30 million cells unstimulated in flask or 10” plates.

After 16h

4. Add 10um MG-132 to restimulated and unstim cells (1:1000 from 10mM MG-132 stock). Wait 4 hours

3. Harvest cells (restimulated cells may need to be scraped off) into falcons, spin down

4. Wash with 1ml cold PBS and transfer into eppendorf, spin down. Remove almost all PBS and mix up the pellet with the remaining PBS.

This protocol describes how to stimulate mouse NK cells in vitro with plate-bound antibodies against NK cell receptors

This protocol describes how to isolate mouse NK cells from spleen using a combination of positive and negative selection techniques