Cd3 fitc

Anesthetized mice were fully perfused with PBS and 4% PFA (paraformaldehyde) and the brains were isolated and fixed with PFA. Before staining, the brain slices (10 μm thick) were washed with PBS three times and then incubated for 30 min in PBS containing 0.3% Triton X-100 and 3% serum. Later, the slices were incubated with primary antibodies for 12 h: rabbit anti-ICAM1 (1:100, Abcam) or mouse anti-A_2AR (1:300, Wako). The sections were then washed with PBS and incubated for 2 h at room temperature with Alexa Fluor 488 antibodies (Abcam; 1:400). The slices were then washed and mounted on slides with VECTASHIELD mounting media (containing DAPI). For CD3 staining, the isolated CP tissues were fixed with 2.5% PFA for 30 min and then transferred to PBS. The following protocol was similar to the above procedure with rat anti-CD3 (1:100, CD3-FITC, Abcam). Images were acquired with a confocal microscope (LSM880, Zeiss). As described earlier [16 (link)], tissue slices were stained with hematoxylin–eosin.

The collected PBMCs were centrifuged at 350 g for 5 min, and the supernatant was discarded. The precipitate was suspended in 300 μL FBS Buffer (BD, USA) and centrifuged; this step was repeated once. Next, 4% paraformaldehyde was fixed and washed 3 times. Subsequently, cells were suspended with 100 μl FBS Buffer containing 2 μl CD3-FITC (Abcam, UK,ab34722), 10 μl CD4-Percp/cy5.5 (BD, USA,561474), and 10 μl CD8-PE (Abcam, UK,ab22548). Next, the cells were incubated at room temperature and protected from the light for 15 min, washed twice, and then, suspended with 100 µL FBS Buffer for on-board testing. Flow cytometry was performed using the CytoFLEX SRT system (Beckman Coulter, USA).

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).