Fitc anti granzyme b

Phorbol myristate acetate (50 ng/ml) and ionomycin (1μg/ml, Sigma) were used to stimulate post-cultured cells. To avoid the secretion of the induced cytokine into the suspension, 2μM GolgiStop with monensin (BD pharmingen) was added. After cultured at 37°C for 4 hours, we harvested and labeled the cells by PE-anti-iNKT monoclonal antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) at room temperature for 20 minutes. The cells then were washed with PBS once. The cells were fixed and permeabilized with 250μl solution at 4°C for 20 minutes, and then washed once with wash buffer. We stained the cells with FITC-anti-IFN-γ, FITC-anti-IL-4, FITC-anti-Granzyme B, and FITC-anti-Perforin (BD pharmingen). Later, the cells were washed once with PBS again, and we analyzed the samples by flow cytometry. Positive expression was shown as percentages.

Freshly isolated PBMCs were stimulated with PAM and IL-2 for 12 days and the cells were cocultured with, or without, allogeneic irradiated UC-MSCs at cell ratios of 5 : 1 or 80 : 1 for 48 hours. During the last 4-hour culture, the cells were treated with Brefeldin A. Subsequently, the cells were harvested, stained with APC-cy7-anti-CD3 and APC-anti-TCRγδ, fixed, permeabilized, and intracellularly stained with FITC-anti-IFNγ, PE-anti-TNFα, PE-anti-IL-10, PE-anti-perforin, or FITC-anti-granzyme B (BD Biosciences) to examine the frequency of cytokine-expressing Vγ9Vδ2 T cells, respectively.