Hypotonic lysis buffer

Peripheral blood (PB) and bone marrow (BM) samples were collected from all groups of mice [6 (link), 22 (link)]. Briefly, a 50 µl of PB was withdrawn from retro-orbital plexus or tail-vein injection in EDTA-coated microvette tubes (Sarstedt Inc., Newton, NC, USA) for hematologic analysis. The blood samples were run on HemaVet 950FS hematology analyzer (Drew Scientific Inc., Oxford, CT, USA) within 2 h of collection. PB was also collected from the posterior vena cava (with a 25-gauge needle and 1-ml syringe containing 250 U heparin). The red blood cells (RBCs) were lysed by hypotonic lysis buffer (BD Biosciences, CA, USA) and PB mononuclear cells (PBMNCs) were obtained by centrifugation. BM mononuclear cells (BMMNCs) were also flushed from tibias and femurs. RBCs present in BM were lysed, washed, and BMMNCs were resuspended in PBS or 2% FBS containing RPMI media as necessary.

Spleens were isolated 17 days after inoculation and mashed using 70 μm filters. Splenocytes were harvested after depletion of RBCs by the hypotonic lysis buffer (BD Biosciences). CD8α-positive splenocytes were isolated by positive selection with anti-CD8α microbeads (Miltenyi Biotec) according to the manufacturer's protocol. The purity of CD8α+ T cells were analyzed with a combination of antibodies specific for CD8α-FITC and CD3e-PerCP-cy5.5 (all purchased from eBioscience, San Diego, CA, USA) by flow cytometry. Then, 2×10⁶ CD8α-positive splenocytes were co-cultured with prepared 5×10⁵ BM-DCs pulsed with 30 μg/ml murine GPC3 proteins (Sino Biological, Inc.) for restimulation. Seven days later, the suspensions containing 4×10⁵ CD8α+ T cells in T-cell medium were added to each well in ELISPOT 96-well plates and stimulated in triplicate with 30 μg/ml GPC3 protein at 37°C for 24 h. The detection of GPC3-specific T cells producing IFN-γ was performed using an ELISPOT kit (BD Biosciences) according to the manufacturer's protocol. Lastly, the plates were dried at normal temperature and the spots were counted with an ELISPOT Reader (CTL Limited) by capturing the images of individual wells. Spot-forming cells were defined as the average number of spots per 4×10⁵ CD8α+ T cells from triplicate wells.