Pe conjugated anti dykddddk

M10 CAR-T cells (5 × 10⁵) were harvested, washed, and stained with PE-conjugated anti-DYKDDDDK (Biolegend #637310) and AF488-conjugated Hu CXCR5 antibody (BD Biosciences #558112) at 4 °C for 30 min. Data were acquired with a BD LSRFortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software.

For tumor cells expressing CD19 and/or PD-L1, 5 × 10⁵ tumor cells were harvested and washed twice with FACS buffer (1× PBS containing 2% FBS). Then, tumor cells were stained with 0.5 μL of APC/Cy7-conjugated mouse anti-human CD19 (BD Pharmingen #557791) or 0.5 μL of APC-conjugated mouse anti-human PD-L1 (eBioscience #17–5983-73) at 4 °C for 30 min, washed with FACS buffer twice, and resuspended in FACS buffer for assessment. Additionally, 5 × 10⁵ washed tumor cells were incubated with 2 μg/mL of trastuzumab prepared in our laboratory at 4 °C for 30 min, washed twice with FACS buffer, and further stained with 0.5 μL of PE-conjugated anti-human IgG Fc (Biolegend #409304) at 4 °C for 30 min, washed with FACS buffer twice, and then resuspended in FACS buffer to detect HER2. To detect the HER2 CAR, PD-L1 CAR or PD-L1 CCR presented on the T cells surface, T cells were stained with Alexa Fluor 647-conjugated anti-Myc tag (CST #2233S) or PE-conjugated anti-DYKDDDDK (Biolegend #637310). T cells (5 × 10⁵) were harvested and washed twice with FACS buffer. For Myc or Flag tag staining, T cells were stained with 0.5 μL of Alexa Fluor 647-conjugated anti-Myc tag or PE-anti-DYKDDDDK at 4 °C for 30 min, washed twice with FACS buffer, and then resuspended in FACS buffer for detection. Fluorescence was assayed using a BD LSRFortessa, and all FACS data were analyzed with FlowJo V10 software.