In vitro proliferation assay using mitogen-stimulated PBMC

YT Yoshiyuki Tago
CK Chiho Kobayashi
MO Mineko Ogura
JW Jutaro Wada
SY Sho Yamaguchi
TY Takashi Yamaguchi
MH Masahiro Hayashi
TN Tomoyuki Nakaishi
HK Hiroshi Kubo
YU Yasuyoshi Ueda
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MSC were seeded at a density of 0.6‒6 × 104 cells/mL in 24-well plates. After 24 h, the cells were incubated with 10 μg/mL mitomycin C (Fujifilm) for 2 h at 37 °C to inhibit cell proliferation. Next, 6 × 105 PBMC/mL were added directly or indirectly using a transwell system (pore size, 0.4 μm; Corning, NY, USA) at a PBMC/MSC ratio of 10:1 or 100:1. The lymphocytes in PBMC were stimulated with 4 μg/mL PHA (Fujifilm) and 100 U/mL IL-2 (PeproTech, NJ, USA). Anti-human antibodies against PD-L1 and PD-L2 (AF156 and AF1224; R&D Systems, MN, USA) were used at 250 or 500 ng/mL to inhibit ligand function. PHA/IL-2-stimulated PBMC and MSC were cocultured for 3 days.

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