Anti human cd4 pe cy7

Anti-human CD4-PE (cat 300508) was from BioLegend. Anti-human CD83-FITC (cat 556910), anti-human HLA-ABC-PE (cat 555553), anti-human HLA-DR-PE (cat 555812), anti-human CD80-FITC (cat 557226), anti-human CD86-APC (cat 555660), anti-human CD25-PE (cat 555432), anti-human CD127-FITC (cat 561697), Anti-human CD4-PE-Cy7 (BD) (cat 348809), anti-TNF-PE-Cy7 (cat 557647), anti-human IL-2-PE (cat 559334), anti-human CD3-APC-H7 (cat 560176), anti-human CD4-CF594 (cat 5562281), anti-human CD4-PB (pacific blue), anti-human CD8-BV605 (cat 564116), anti-human CD8-PerCP-Cy5.5 (cat 341050), anti-human IFN-γ–AF700 (cat 557995), anti-human CD35-PE-Cy7 (cat 557741), anti-human IL-10 (cat 554707) were from Becton Dickinson, Pharmigen. Anti-human Foxp3-APC (cat 17-4776-41) was from eBioscience. Anti-human CD14-PE (cat A07764) was from Beckman Coulter. Anti-human CD45RO-APC/Cy7 (cat 304227), anti-human CD45RA-PE (cat 304205), anti-human CD3-APC (cat 300411), and anti-human CD40-APC (cat 313008) were from BioLegend). Anti-human IL-10-BV421(cat 501421), anti-human CD45RA-BV711 (cat 304137), anti-human CD127-BV711 (cat 351327) from Biolegend, anti-human CD45RO-ECD (cat IM2712) (BC), anti-human FoxP3-PE (cat 12-4777-42), from eBiosciences.

Non-specific sites of 2.5 × 10⁵ cells were blocked with rabbit immunoglobulins G (IgG, Sigma-Aldrich). Then, the cells were incubated with fluorochrome-conjugated monoclonal Ab (mAb) and isotype-matched negative controls for 20 min at 4 °C. The following mAbs were used: anti-human CD3 APC-Vio770 (Miltenyi Biotec, Bergisch Gladbach, Germany), anti-human CD4 PE-Cy7 and anti-human CD62L FITC allophycocyanin (both from BD Pharmingen, San Diego, CA) and isotype-matched mAb (Miltenyi Biotec). After staining, the cells were washed and re-suspended in PBS (Sigma-Aldrich) supplemented with 0.2 % bovine serum albumin and 0.01 % sodium azide (both from Sigma-Aldrich). The samples were collected and analysed using a CyAn ADP, running Summit 4.3 analysis software (Beckman Coulter, Brea, CA, USA). The living cells identified by propidium iodide (Sigma-Aldrich) exclusion were gated according to their light scatter properties to exclude cell debris. Quality experiments have been performed (Additional file 1: Figure S1) in order to evaluate whether the freeze-thawing cycle could affect the quantification of CD62L protein expression and to check the reproducibility of the flow cytometric procedure and the stability of the expression of CD62L on CD4⁺ T cells in frozen samples.

Tonsillar MNCs were treated with Brefeldin A (eBiosciences) for 4 h before harvesting cells in order to block cytokine secretion. MNCs were stained with fluorescence labeled anti-human CD4, followed by fixation and permeabilization and staining intracellularly with fluorescence labeled anti-human Foxp3, IL-17A, IFNγ, IL-10, RORγt, and Ki67 in different combinations. Stained cells were acquired on BD Celesta or BD Canto II and data was analyzed by Flowjo. Anti-human CD4-PECy7, IL-17A-Alexa Fluor 647, Foxp3-Alexa Fluor 488, IL-10-PE, and RORγt-PE FACS antibodies were from BD Biosciences. Other antibodies were purchased from BioLegend.