1. A B C Day 0 Side scattering Forward scattering Day 0 Day 10
Online Resource 2. Gating strategy for flow cytometry.
Dot plots of forward and side scattering showing gating for (A) Fig. 1A, (B) Fig. 2 day 0, and (C) Fig. 2 day 10.
Peripheral blood samples were obtained from patients with mammary carcinoma after institutional review board approval and with written informed consent. PBMC were purified by Ficoll-PaqueTM PLUS (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) gradient centrifugation. After being washed two times with PBS, the cells were resuspended at 1×107 cells/6 mL of modified Yssel’s medium [1] supplemented with 10% human AB serum (Cosmobio., Co., Ltd., Koto-ku, Tokyo, Japan). For flow cytometric analysis, PBMC were plated out at 2×105 cells/50 mL in a 96-well plate (Corning Incorporated, Corning, NY). The cells were then treated with 3 mL of fluorescein isothiocyanate (FITC)-conjugated anti-TCR Vd1 mAb (Beckman Coulter Inc., Fullerton, CA), FITC-conjugated anti-TCR Vd2 mAb (Beckman Coulter), and/or phycoerythrin (PE)-conjugated anti-CD3 mAb (BD Biosciences, San Diego, CA) on ice for 30 min. After being washed three times with PBS, the cells were resuspended in 200 mL of 1% paraformaldehyde in PBS and analyzed by means of flow cytometry.
1.Yssel, H., De Vries, J.E. Koken, M., Van Blitterswijk, W. and Spits,H., Serum-free medium for generation and propagation of functional human cytotoxic and helper T cell clones. J. Immunol. Methods :