Fitc conjugated anti human cd3

The purity of isolated T cells was confirmed using APC-conjugated anti-human CD3 (Clone: UCHT1, BioLegend). PE-conjugated anti-human mesothelin (Clone: #420411, R&D Systems) was used to detect mesothelin expression. FITC-conjugated anti-human CD3 (Clone: HIT3a, BioLegend), PE-conjugated anti-human CD279 (PD-1) (Clone: EH12.2H7, BioLegend), and APC-conjugated anti-human CD366 (Tim-3) (Clone: F38-2E2, BioLegend) antibodies were used to measure the expression of exhaustion markers. For proliferation assays, cells were loaded with CellTrace™ CFSE (Life Technologies, #C34554) according to manufacturer’s instructions, and T cells were detected using PerCP-conjugated anti-human CD3 antibody (Clone: HIT3a, BioLegend). Data were collected using a BD FACSCalibur (BD Biosciences) and analyzed with FlowJo software (v10.6). All assays were performed in duplicate and repeated two to three times.

Cells were labeled with monoclonal antibodies for 30 min at 4 °C in FACS sorting buffer. The following antibodies were used: BV421‐conjugated anti‐human CD45 (BD Biosciences, 563879), FITC‐conjugated anti‐human CD3 (Biolegend, 300306), APC‐H7‐conjugated anti‐human CD4 (BD Biosciences, 560158), Percp‐cy5.5‐conjugated anti‐human CD8 (Biolegend, 344710). After staining, cells were washed once and resuspended in FACS sorting buffer. Cell sorting was performed using BD FACS Aria III. All samples were gated based on forward and side scatters, followed by exclusion of doublets. Expression levels of CD45, CD3, CD4, and CD8 were gated by their negative controls of unstained cells and positive controls of cells stained by each antibody. CD45⁺CD3⁺ T cells were sorted into 1.5 mL tubes containing 300 µL ice‐cold PBS with 1% BSA. The abundance of the relevant cell populations determined by testing the sorted cells again by FACS, reached more than 99%. Data analysis was performed using FlowJo V10 software (http://www.flowjo.com).

CD123 expression was detected using a PE-conjugated anti-human CD123 antibody (Clone 6H6, Catalog No. 306006, BioLegend; San Diego, CA, USA). Samples were analyzed on a NovoCyte 3000 Flow Cytometer. CD123 cell surface expression was quantitated with the use of BD Quantibrite PE Phycoreythrin Fluorescence Quantitation kit (Catalog No. 340495, BD Biosciences, San Jose, CA, USA) following the manufacturer’s protocol.
Peripheral blood was stained with APC-conjugated anti-mouse CD45 (BioLegend Catalog No. 103112), Pacific blue conjugated anti-human CD45 (BioLegend Catalog No. 304029) and FITC conjugated anti-human CD3 (BioLegend Catalog No. 317306) antibodies following incubation with human BD Fc block (BD Biosciences, Catalog No. 564219) to prevent non-specific antibody binding.