Fitc conjugated anti mouse antibody

HLA-A*0201 surface expression as well as intracellular Melan-A/MART-1 antigen expression was determined by flow cytometry using a FITC-conjugated mAb BB7.2 for HLA-*0201 and a Melan-A/MART-1 antibody (NovoCastra) followed by staining with FITC-conjugated anti-mouse antibody (BD Biosciences) for Melan-A/MART-1. Cells were analyzed on a Becton Dickinson FACSCalibur with Cell Quest software (BD Biosciences).

Cells were harvested using RIPA buffer for Western blot, and 40 μg of total protein were run on 12 % gels followed by wet transfer at 25 V overnight at room temperature. The blots were then blocked with 10 % milk, followed by incubation with primary antibody and HRP-conjugated secondary antibody. Protein bands were detected with ECL (Perkin Elmer, Cat.no. NEL105001EA). 8x10⁵ cells were plated for flow cytometry, treated and harvested for fixation and permeabilization (BD Pharmingen, Cat. no. 560409). Cells were blocked with blocking serum, incubated with HDAC 1 antibody, washed, incubated with secondary FITC-conjugated anti-mouse antibody (BD bioscience, Cat.no. 554001) and finally stained with propidium iodide for cell cycle analysis. Cells were run on a LSR Fortessa, and results were analyzed using FCS Express software.