Facs calibur with sorter

To identify the percentage of Tregs in peripheral circulation, the flow cytometric procedure of Fujimoto et al. was followed with slight modification.^{9 (link)} In brief, a 25 μl aliquot of whole blood was diluted with 75 μl of phosphate buffered saline (PBS, pH 7.4) and the diluted blood samples were incubated with 10 μl each of FITC anti-human CD4 and PE anti-human CD25 monoclonal antibodies (BioLegend, San Diego, CA, USA) and isotype-matched negative controls for 30 min in the dark at room temperature. The erythrocytes were then lysed by incubating the samples with 2 ml of RBC lysing solution (BD biosciences, CA, USA) for 5 min at room temperature. Thereafter, the cells were fixed with 0.5% paraformaldehyde and 15,000 events were acquired and analyzed in a flow cytometer (FACS Calibur with sorter, BD biosciences, San Jose, CA, USA). Cells were identified from their characteristic forward and side scatter profile on dot plots and gated. Data acquisition and analysis of FL-1 (FITC) and FL-2 (PE) were done using Cell Quest software (BD biosciences, USA). The relative proportion of Tregs was calculated from the statistical package of the Cell Quest software from quadrant gate setting.

2×10⁴ B16/F10 melanoma cells both for treated and control were harvested, washed with PBS, resuspended in sterile PBS. Cells were fixed in ice cold 100% methanol −20°C for 1 h. The cells were centrifuged at 5,000 rpm for 10 min and the cell pellets were suspended in 1 ml of hypotonic buffer (0.5% Triton X-100 in PBS and 0.5 mg/ml RNase), and incubated at 37°C for 30 min. Subsequently, propidium iodide solution (50 mg/ml) was added, and the mixture was allowed to stand for 1 h in darkness. Fluorescence that emitted from the propidium iodide-DNA complex was quantitated after excitation of the fluorescent dye by (FACS Calibur with sorter, BD Biosciences, San Jose, CA) using Cell Quest software (BD Biosciences, San Jose, CA) equipped with a 488 nm argon laser and a 525±10 nm band pass emission filter. Fluorescence was captured on a FL2H channel with linear amplification.