Percp conjugated anti cd4

On the seventh day after the injection of SCC7 cells, the mice were sacrificed, and the tumour tissues were extracted to make a single‐cell suspension. The mouse TILs were isolated from the tumours using the tumour lymphocyte infiltration kit (Solarbio, Beijing, China), stained with APC‐conjugated anti‐CD3, PE‐conjugated anti‐CD8 and PerCP‐conjugated anti‐CD4 (Biolegend, San Diego, CA, USA), and detected using flow cytometry.
For the apoptosis assay, SCC7 cells were harvested and detected using Annexin V‐PE/7‐AAD staining assay and flow cytometry.

Cells of interest were stained with different combinations of the following antibodies: FITC anti-CD3, brilliant violet 510 (BV510) anti-CD4, peridinin chlorophyll (PerCP)-conjugated anti-CD4, APC anti-CD8, APC/Cy7 anti-CD8, APC/Cy7 anti-CD45, PE anti-CD45, PE anti-CD45RO, APC/Cy7 anti-CD62L (all BioLegend) and PE-conjugated HLA-A*02:01/CMVpp65p-specific dextramer (Immudex, Copenhagen, Denmark). Surface staining was performed at room temperature for 20 min in the dark and washed with PBS (Lonza, Vervies, Belgium) with 0.1% human AB serum (c.c. pro). 7-amino-actinomycin D (7-AAD; BD Biosciences) was applied prior to flow cytometric analysis to exclude dead cells. All samples were analyzed by multicolor flow cytometry (FACS Canto II, FACSDiva V8.1.2 software, FlowJo_v10.7.1 software; all BD Biosciences). Gates were set based on the forward scatter versus side scatter properties of lymphocytes. At least 30,000 events were acquired in the CD3⁺ gate.

The following anti-human monoclonal antibodies (mAbs) were used for flow cytometry: fluorescein isothiocyanate (FITC)-conjugated anti-HLA-DR; phycoerythrin (PE)-conjugated anti-CD86; and allophycocyanin (APC)-conjugated anti-CD83 (Miltenyi Biotec). APC-conjugated anti-CD3; and Alexa Fluor 488-conjugated anti-IFN-γ (BD Pharmigen). PE-conjugated anti-IL-10; Alexa Fluor 488-conjugated anti-IL-17A; peridinin-chlorophyll-protein (PerCP)-conjugated anti-CD4; Alexa Fluor 488-conjugated anti-FOXP3; PE-conjugated anti-CD127; and APC-conjugated anti-CD25 (Biolegend). Cells were washed with PBS/EDTA 2 mM/0.5% BSA and stained for 15 min at room temperature in the darkness. For analysis of FOXP3 expression in human T cells primed with hmoDCs, cells were first subjected to surface staining with anti-human CD127-PE, CD4-PerCP, and CD25-APC antibodies. After fixation and permeabilization, cells were stained with anti-human FOXP3-Alexa Fluor 488, according to manufacturer’s recommendations. For each staining, the corresponding isotype controls (IgG2A-FITC, IgG1-Alexa Fluor 488, IgG1-PE, IgG2A-PerCP, or IgG1-APC) were also assayed. Flow cytometry analysis was performed in a FACSCalibur in the Cytometry and Fluorescence Microscopy Unit at Complutense University of Madrid.