Anti human cd4 apc

Fresh samples, including tumor tissues (n = 33) and peritumor tissues (n = 33) from NSCLC patients were collected from Weifang Second People’s Hospital. The peritumor area was defined with a radius of at least 2 cm from the tumor periphery.
Single-cell suspensions were prepared from tumor tissue. Then samples were stained with anti-human VISTA (PE, clone: MIH65, BD Pharmingen), anti-human CD45 (PC5.5, clone: J33, Beckman Coulter), anti-human CD4 (APC, clone: 13B8.2, Beckman Coulter), anti-human CD8 (APC-A750, clone: B9.11, Beckman Coulter) for 30 minutes at 4° C after being lysed with erythrocyte lysis buffer. Stained cells were washed and resuspended in phosphate-buffered saline. Flow cytometry data were analyzed by FlowJo software (BD, Bioscience).

Th2-polarized CD4⁺ T cells were platted in a density of 1 × 10⁵ cells per well. Fc receptors were blocked by incubation in a buffer containing 10 µg/mL heat-aggravated human immunoglobulin G (IgG; Sigma, Deisenhofen, Germany). An extracellular epitope of CRTH2 was stained with anti-human CRTH2-PE (mouse monoclonal IgG2a, 1 µg/100 µL; Thermo Fisher) the respective isotype control IgG2a was tested in parallel. CD4⁺ T cells were stained with anti-human CD4-APC (1 µg/100 µL; Beckman Coulter, Krefeld, Germany), the respective isotype control IgG1 was tested in parallel. To assess cell viability, Th2-polarized CD4⁺ T cells were incubated with Fixable Viability Dye eFluor^® 660 (BD Bioscience) for 30 min before staining.
After washing with PBS/BSA 1%, sample acquisition was performed by flow cytometry (FACS Calibur, Becton Dickinson, Heidelberg, Germany) and the percentage of gated cells was calculated using the Flow Jow Pro software (Becton Dickinson).