Lymphocyte separation medium

Animals were immobilized with (10 mg/kg) ketamine-HCl intramuscularly (IM) prior to all sample collections. Complete blood counts (CBC) were performed at each blood collection. Infant EDTA blood samples were collected longitudinally starting at birth and subsequently every month for one year. Plasma was removed from whole blood by centrifugation and stored at −80°C for antibody measurement. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood via density centrifugation using Lymphocyte Separation Medium (LSM) (MP Biomedicals, Solon, OH) or Accu-Paque Lymphocyte separation media (Accurate Chemical & Scientific Corp., Westbury, NY) as described [51 (link)-53 (link)]. Due to the low blood volume that could be collected from infant macaques, and therefore relatively low cell counts, infant PBMC were used immediately for functional analyses. PBMC from adolescent and adult macaques were cryopreserved for functional assays and batch-analysis.

Calcein-AM or calcein-red: Thermo Fisher Scientific (Burlington, ON, Canada); Caspase 1 inhibitor (Ac-YVAD-CMK): Sigma (Oakville, ON, Canada); Caspase 3 inhibitor (Z-DEVD-FMK): APExBIO (Boston, MA, USA); Caspase 8 inhibitor (Z-IETD-FMK): MBL International Corporation (Woburn, MA, USA); Caspase 9 inhibitor (Z-LEHD-FMK): MBL International Corporation (Woburn, MA, USA); CD31 Dynabeads: Life Technologies AS (Oslo, MN, USA); Endothelial Cell Growth Medium-2 (EGM-2): Lonza (Walkersville, MD, USA); Evans blue (EB)-labeled bovine serum albumin (BSA): Sigma (Oakville, ON, Canada); fluoroisothiocyanate (FITC) or Alexa Fluor 647-labeled Annexin V: BioLegend (San Diego, CA, USA); FITC-labeled dextran: Sigma (Oakville, ON, Canada); Human TNFα, IL1β, IFNγ: PeproTech (Rocky Hill, NJ, USA); Lymphocyte Separation Medium (LSM): MP Biomedical (Canada); Pan-caspase inhibitor (Q-VD-OPh hydrate): APExBIO (Boston, MA, USA); Sulforhodamine (SR) FLICA Poly Caspase Assay Kit: Immunohistochemistry Technologies (Bloomington, MN, USA); terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) In Situ Cell Death Detection kit: Roche (Laval, QC, Canada); Type II collagenase: Worthington Biochemical Corporation (Lakewood, NJ, USA).

Peripheral blood mononuclear cells (PBMCs) from healthy donor blood were isolated using Lymphocyte Separation Medium (MP Biomedicals) (NIH protocol 99-H-0050 and 2006/229-31/3). NK cells were isolated by magnetic bead separation; either CD3 depleted and CD56 selected (Miltenyi) or by using an NK cell isolation kit (Miltenyi). NK cells were expanded for 14–18 days in G-Rex flasks (Wilson Wolf Manufacturing) with irradiated human EBV-LCL feeder cells at a ratio of one NK cell to 10 feeder cells (1:10 ratio). Cells were cultured in X-vivo 20 media (Lonza) containing 10% human AB serum (Sigma), 1% Glutamax (Gibco), and 500 U/ml IL-2. Fresh media was supplied to cells starting on day 5 of expansion, and then every 48 h until the cells were harvested for use in experiments. Cryopreserved WHIM PBMCs (NIH protocols NCT05-I-0213; NCT14-I-0185) were generously provided by Dr. David McDermott through an NIH Material Transfer Agreement in accordance with NIH human subject research policies. K562 and MOLM14 cell lines were obtained from ATCC. MM.1S was kindly provided by Dr. Irene Ghobrial at the Dana-Farber Cancer Institute. SMI-LCLs were established in the NHLBI/NIH by the Childs' lab. Cell lines were propagated in RPMI 1640 supplemented with 10% heat-inactivated FBS (Sigma-Aldrich).