Apc labeled anti cd4

Inguinal dLNs were collected 7 days after immunization. Single LN cells were stained with FITC-labeled anti-CD3 (Biolegend, Cat# 100203), APC-labeled anti-CD4 (Biolegend, Cat# 100411), PE-labeled anti-PD1 (Biolegend, Cat# 135205), and PECy7-labeled anti-CXCR5 antibodies (Biolegend, Cat# 145515) to analyze Tfh cells or Alexa Fluor® 700-labeled anti-CD45 (Biolegend, Cat# 103127), FITC-labeled anti-B220 (Biolegend, Cat# 103205), APC-labeled anti-GL7 (Biolegend, Cat# 144617), and PE-labeled anti-Fas antibodies (Biolegend, Cat# 152607) to analyze GC B cells.

Tumor tissues were harvested and treated with 1 mg/mL collagenase I (Gibco) for 1 h and ground using the rubber end of a syringe. Cells were filtered through nylon mesh filters and washed with PBS. Cells were further stained with corresponding fluorochrome-conjugated antibodies. To analyze the T-cell subsets in draining tumor, single-cell suspensions prepared from these samples were examined by flow cytometry. The following primary antibodies were used: PerCP-Cyanine5.5-labeled anti-CD3 (BioLegend, catalog: 100328), APC-labeled anti-CD4 (BioLegend, catalog: 100412), PE-Cy7-labeled anti-IFN-γ (BioLegend, catalog: 505826), and FITC-labeled anti-Ki67 (eBioscience, catalog: 11-5698-82). Antibodies were used at a dilution of 1:200. Data analysis was acquired on a CytoFLEX and analyzed using CytExpert (Beckman Coulter, USA) and FlowJo (TreeStar, USA) software.