7 aminoactinomycin d 7 aad solution

The expression of HER2 on 4T1-Luc-HER2 cells was detected using allophycocyanin (APC) labeled anti-human CD340 (erbB2/HER2) antibody, with APC mouse IgG1κ (both from BioLegend, San Diego, CA, USA) used as an isotype control. The expression of PDL1 on 4T1-Luc-HER2 cells was determined using PE/Cy7 anti-mouse CD274 (PDL1) antibody (BioLegend), with PE/Cy7 Rat IgG2bκ (BioLegend) used as an isotype control. The expression of CD3 in the activated mouse T cells was evaluated using APC anti-mouse CD3ε (BioLegend), with APC Armenian Hamster IgG (BioLegend) used as an isotype control. 7-aminoactinomycin D (7-AAD) solution (BioLegend) was used to determine the percentages of viable CAR-T cells. The expression of PD1 on anti-HER2 CAR-T cells was examined using PE-conjugated anti-mouse PD1 antibody (BD Biosciences), with PE mouse IgG2aκ (BD Biosciences) as an isotype control.

To measure CAR expression, 0.25 million cells were suspended in 100 µL of buffer (Phosphate-buffered saline (PBS) containing 2 mM Ethylenediaminetetraacetic acid (EDTA) pH 8 and 0.5% Bovine serum albumin (BSA)) and incubated on ice with 1 µL of human serum (Jackson Immunoresearch, West Grove, PA, USA) for 10 min. Then, 0.3 µg of biotinylated human DCLK1 protein (AcroBiosystems, Newark, DE, USA) was added and the cells were incubated on ice for 30 min. Cells were rinsed with 3 mL of buffer and suspended in 100 µL of buffer. Next, 1 µL of phycoerythrin (PE)-conjugated streptavidin (BD Biosciences, San Jose, CA, USA), 1 µL of allophycocyanin (APC)-labeled anti-CD3 (BioLegend, San Diego, CA, USA) and 2 µL of 7-aminoactinomycin D (7-AAD) solution (BioLegend) were added to the cells and incubated on ice for 30 min [39 (link),42 (link)]. The cells were rinsed with 3 mL of buffer and suspended in buffer and acquired on a FACSCalibur (BD Biosciences). Cells were analyzed first for light scatter versus 7-AAD staining, and the 7-AAD⁻ live gated cells were plotted for anti-CD3 staining versus DCLK1 protein staining [39 (link),42 (link)].