Ac devd cho

The inhibitory effects on tumor cell viability observed following treatment with the MW hyperthermia device or water bath were determined by CCK-8 assay. The NSCLC cells, H460, PC-9 and H1975, were seeded in 96-well plates at 1×10⁴ cells/well. The cells were exposed to different temperatures (moderate hyperthermia) for 60 or 90 min. In the negative control group, the cells were incubated at 37°C in a CO₂ incubator instead of MW irradiation or the water bath, and then incubated in a CO₂ incubator. After 6, 12 or 24 h of treatment, 10 µl of CCK-8 solution (MedChem Express, Princeton, NJ, USA) were added to each well, and the cells were cultured for a further 1–3 h at 37°C. Cell viability in each group was measured at 450 nm using a Multiskan Spectrum spectrophotometer (Thermo Fisher Scientific, Rockford, IL, USA). The cells were also pre-treated with or without the caspase-3 inhibitor, Ac-DEVD-CHO (Selleckchem, Houston, TX, USA), for 3 h prior to MW hyperthermia (43°C for 90 min) for 24 h. Cell viability in each group was then measured.

1 d before immunization, OT-I T cells were prepared from pooled cell suspensions from spleens and peripheral LNs as described earlier, and 5 × 10⁵ OT-I T cells in PBS were transferred into recipient mice by intravenous injection. Recipient mice were immunized by subcutaneous injection into the flanks with a dose of 10 µg OVA (Imject Ovalbumin, Thermo Fisher Scientific) + 50 µg poly(I:C) (Sigma-Aldrich) in PBS. For longitudinal studies of transferred OT-I populations, blood lymphocytes from retro-orbital bleeds were analyzed by flow cytometry. For terminal analysis of donor OT-I T cell phenotype, total cell suspensions prepared from inguinal LNs were restimulated with 5 µg/ml OVA SIINFEKL peptide (Sigma-Aldrich) for 3–4 h before analysis by flow cytometry. The contribution of LN egress and/or activation-induced cell death on T cell numbers was examined by intraperitoneal injection of 500 µg/kg FTY720 (Cayman Chemical) in a vehicle of 50% (vol/vol) ethanol in PBS and/or 3 mg/kg pan-caspase inhibitor peptide Ac-DEVD-CHO (Selleck Chemical) at indicated time points.