Phytohemagglutinin p

MMCT was performed essentially as previously described (Meguro-Horike and Horike 2015 (link)). For each experiment, six T-25 flasks of A9-14 or A9-15 cells at 80%–90% confluency were treated with 0.075 µg/mL demecolcine (Sigma) for 48 h. On the day of MMCT, the flasks were filled to the neck with prewarmed serum-free F-12 media supplemented with 10 µg/mL cytochalasin B (Sigma). Flasks were submerged in water and centrifuged in 500-mL bottles (Nalgene) at 10,000g for 1 h at 34°C. The microcell pellet was resuspended in serum-free F-12 media and filtered through nitrocellulose membrane filters (8 µm and 5 µm, respectively; Whatman) in 25-mm Swinnex filter holders (Millipore). Microcells were centrifuged at 1500g for 10 min. The pellet was resuspended in 2 mL of serum-free F-12 media containing 200 µg/mL phytohemagglutinin P (Sigma) and applied to a 70% confluent 60-mm dish of hTERT-RPE1 Hyg^−ve cells. Following 30-min agglutination at 37°C, media was removed and membrane fusion was facilitated by addition of 1 mL of PEG 1500 for 2 min. Cells were then extensively washed with serum-free F-12 media. Following 24-h incubation in complete media, MMCT fused cells were selected using 400 µg/mL hygromycin and 20 µg/mL blasticidin. Clones were visible 2–3 wk later.

Peripheral blood mononuclear cells (PBMCs) were separated from EDTA-anticoagulated blood by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare). Freshly isolated PBMCs from four donor MCMs heterozygous for the M1 and M2 MHC haplotypes were pooled and resuspended in RPMI-1640 containing 10% fetal calf serum (FCS; R10) and 5 μg/ml phytohemagglutinin-P (Sigma-Aldrich). The culture was incubated overnight at 37°C in a 5% CO₂ atmosphere. Activated PBMCs were harvested the following morning, washed twice with phosphate-buffered saline (PBS), resuspended in PBS, and loaded into tuberculin syringes. Each macaque was then immunized with up to 4 × 10⁷ activated PBMCs as described previously (29 (link)).