Anti igfbp2

For specific depletion of IGF-axis proteins from FF, 25 μl protein G beads (Santa Cruz, sc-2002) were incubated with 10 μl (0.2 μg/ml) of anti-IGF1 (Santa Cruz, sc-9013), anti-IGF2 (Genetex, GTX60630), anti-IGFBP2 (Santa Cruz, sc-6001), anti-IGFBP6 (Santa Cruz, sc-6007), or anti-PAPP-A1 (Santa Cruz, sc-365226) antibodies at 4 °C for 3 h. The antibody-conjugated beads (24 μl) were each added to 400 μl of FF and rotated overnight at 4 °C. After centrifugation to remove the beads, the supernatants were used for experiments.

EmCaMSC2 and EmCaMSC3 were seeded at a cell density of 1 × 10⁵. After attaching CaMSCs, PBMCs were added and the cells were cocultured at different ratios (1:1, 1:2, 1:4, and 1:8), with or without IFNγ (100 ng/ml) treatment for 3 days. The cell viability was measured by using 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)(2H)-tetrazolium-5-carboxanilide (Biological Industries Ltd., Beit-Haemek, Israel). Briefly, the optical density was measured at 450 nm, and the absorbance at the reference wavelength of 650 nm was subtracted and adjusted with that of the blank control (wells without cells). All of the wells contained anti-CD3 (5 µg/ml) and anti-CD28 (5 µg/ml) antibodies to promote T-cell proliferation, and all experiments were conducted in triplicate. PBMC cells alone (0:1, 0:2, 0:4, and 0:8) were used as the positive control. For blocking or activating experiments, nivolumab (3,000 ng/ml; Opdivo), anti-PD-L1 (3,000 ng/ml; Abcam), recombinant IL-8 (300 ng/ml; ProSpect), anti-IL-8 (3,000 ng/ml; Abcam), anti-IGFBP2 (3,000 ng/ml; Santa Cruz Biotechnology, Dallas, TX, USA), and anti-insulin-like growth factor–binding protein 6 (IGFBP6) (3,000 ng/ml; Santa Cruz Biotechnology) were used.