Human peripheral blood mononuclear cells (PBMCs) from healthy volunteers (20–60 years of age), patients with CD (25–60 years of age), and patients with CD taking 6-MP (25–58 years of age) were isolated using SepMate™ and Lymphoprep™ (Stemcell Technologies, Vancouver, BC, Canada) density gradient medium according to the manufacturer’s protocol. NK cells were purified from PBMCs by negative selection technique using EasySep™ (Stemcell Technologies) human NK cell enrichment kit according to the manufacturer’s protocol. The purity of isolated NK and T cells were confirmed by flow cytometry to be above 90%.
Cultures of NK cells were performed in complete medium composed of RPMI-1640, 10% heat inactivated FBS, 100 IU mL−1 penicillin and 100 μg mL−1 streptomycin, 2 mM L-glutamine, 10 mM HEPES buffer (Cellgro, Manassas, VA), 5 × 10−5 M 2-ME (Sigma-Aldrich, St. Louis, MO), and 1 ng mL−1 (13 IU) recombinant human IL-2 (R&D Systems, Minneapolis, MN) [31 (link)]. NK cells were cultured at 5–10 × 104 cells mL−1 in 96-well plates for 24–72 h. For cell survival and Rac-1 assays, cells were cultured with 5–25 μM of 6-MP (Sigma Aldrich), 5–10 μM of 6-TG (Sigma Aldrich), or 100 μM of Rac1 inhibitor ITX-3 (Sigma Aldrich) to mimic physiologic levels of 6-MP and 6-TG as previously described [26 (link)].
Cultures of NK cells were performed in complete medium composed of RPMI-1640, 10% heat inactivated FBS, 100 IU mL−1 penicillin and 100 μg mL−1 streptomycin, 2 mM L-glutamine, 10 mM HEPES buffer (Cellgro, Manassas, VA), 5 × 10−5 M 2-ME (Sigma-Aldrich, St. Louis, MO), and 1 ng mL−1 (13 IU) recombinant human IL-2 (R&D Systems, Minneapolis, MN) [31 (link)]. NK cells were cultured at 5–10 × 104 cells mL−1 in 96-well plates for 24–72 h. For cell survival and Rac-1 assays, cells were cultured with 5–25 μM of 6-MP (Sigma Aldrich), 5–10 μM of 6-TG (Sigma Aldrich), or 100 μM of Rac1 inhibitor ITX-3 (Sigma Aldrich) to mimic physiologic levels of 6-MP and 6-TG as previously described [26 (link)].