Apc conjugated anti mouse igg

After B16BL6-CAR/E1B55 cells were infected with recombinant adenovirus for two days, infected cells were trypsinized and washed twice with ice-cold PBS. Then cells were incubated with anti-MART1 antibody for 1 h at 4°C. After washing twice with ice-cold PBS, cells were incubated with Allophycocyanin (APC)-conjugated anti-mouse IgG (BD Biosciences, Lincoln Park, NJ, USA) antibody in the dark for 45 min at 4°C. Cells were then washed twice with ice-cold PBS. As a negative control, mouse IgG fluorescence control (BD Biosciences) antibody was used. Finally, cells were suspended again in PBS and analyzed using a flow cytometer. For the detection of various immune cells after combination treatment of MART1 plasmid with Ad^MGshT, the following antibodies were used for staining: CD3-FITC, CD4-PE/Cy7, CD25-APC (Abcam, UK), and CD122-PE/Cy7, CD8a-APC, Foxp3-PE, NK1.1-APC (Biolegend, USA). After adding the appropriate antibody, the cells were incubated in the dark at 4°C for 30 minutes and washed 3 times by centrifugation using ice cold-PBS buffer, and then analyzed on a flow cytometer (Beckman Coulter, Inc., CA, USA). Data were analyzed by using FACSuit Software (BD, CA, USA).

Single-cell suspensions were fixed and permeabilized using BD Cytofix/Cytoperm and stained with mouse anti-Crk (BD Biosciences, 610035) and rabbit anti-CrkL (Santa Cruz Biotechnology, sc-319) antibodies, followed by APC-conjugated anti-mouse IgG (BD Biosciences, 550826) and BV421-conjugated anti-rabbit IgG (BD Biosciences, 565014). Cells were analyzed by flow cytometry using the On-chip Sort (On-chip Biotechnologies).