Anti human cd4 alexa fluor 450

Cell cultures were performed in RPMI 1640 (Sigma Aldrich, UK) containing 10 % FCS (Biosera, UK) supplemented with glutamine/penicillin/streptomycin (Life Technologies, UK). PBMCs (1 × 10⁶/ml) were stimulated for 4 h with PMA (50 ng/ml; Sigma Aldrich, UK) and Ionomycin (500 ng/ml; Sigma Aldrich, UK) in the presence of Brefeldin A (10 μg/ml; Sigma Aldrich, UK). Post stimulation, cells were washed twice with PBS and stained using anti-human CD3 PEcy7 (eBiosciences, UK; clone: UCHT1) and anti-human CD4 Alexa fluor 450 (eBiosciences, UK; clone: RPA-T4) for 20 min in the dark at 4 °C. Cells were washed and fixed with Reagent A (Fix and Perm kit, Invitrogen, UK) for 30 min in the dark at room temperature. Post incubation, cells were washed and re-suspended in Reagent B (Fix and Perm kit, Invitrogen, UK) and anti-human Alexa fluor 647 IL10 antibody (clone: JES3-9D7) was added to cells that were incubated in the dark at room temperature for 30 min. After washing the cells were resuspended in PBS and analysed on a Cyan ™ ADP (Dako Ltd, UK) and the percentage of CD3⁺ CD4⁺ IL10⁺ T cells and IL10 expression levels (MFI value) by CD4 T cells was recorded.

Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation using Ficoll-Paque™ PLUS (GE Healthcare, Sweden). Isolated PBMCs were resuspended in phosphate buffered saline (PBS) at a concentration of 1 × 10⁶/ml and were stained with anti-human CD3-PEcy7 (eBiosciences, UK; clone: UCHT1), anti-human CD4 Alexa fluor 450 (eBiosciences, UK; clone: RPA-T4) and anti-human CD25 APC (Biolegend, UK; clone: BC96) antibodies for 20 min in the dark at 4 °C. Post incubation, cells were washed with PBS and re-suspended in Foxp3 Fix Perm Working solution (eBiosciences, UK) and incubated for 30 min in the dark at room temperature. Post incubation, cells were washed and resuspended in Foxp3 Permeabilization buffer (eBiosciences, UK) and stained with anti-human Foxp3 PE antibody (eBiosciences, UK) for 30 min in the dark at room temperature. Finally, the cells were washed and resuspended in PBS for flow cytometric analysis using a Cyan ™ ADP (Dako Ltd, UK). The percentage of CD3⁺ CD4⁺ CD25⁺ Foxp3⁺ T cells were recorded.

PBMC cultures were performed in complete RPMI 1640 (Sigma Aldrich, UK) containing 10% FCS (Biosera, UK) supplemented with glutamine/penicillin/streptomycin (Life Technologies, UK). PBMCs were stimulated for 4 hr with PMA (50 ng/ml; Sigma Aldrich, UK) and Ionomycin (500 ng/ml; Sigma Aldrich, UK) in the presence of Brefeldin A (10 μg/ml; Sigma Aldrich, UK). Post-stimulation, cells were washed twice with PBS and stained using anti-human CD3 PEcy7 (eBiosciences, UK; clone: UCHT1) and anti-human CD4 Alexa fluor 450 (eBiosciences, UK; clone: RPA-T4) for 20 min in the dark at 4°C. Cells were washed and fixed with 50 μl Reagent A (Fix and Perm kit, Invitrogen, UK) for 30 minutes in the dark at room temperature. Post incubation, cells were washed and re-suspended in 50 μl Reagent B (Fix and Perm kit, Invitrogen, UK) and anti-human TNFα PE (eBiosciences, UK; clone: MAb11) or anti-human IL6 APC (eBiosciences, UK; clone: MQ213A5) or anti-human IL17A Alexa fluor 647 (Biolegend, UK; clone:ebio64Dec13) or anti-human IFNγ APC (Bio legend, UK ;clone: AS.B3) and anti-human IL4 PE (eBiosciences, UK; clone:8D4-8) was added and samples were incubated in the dark at room temperature for 30 minutes. After washing the cells were analysed on a Cyan™ ADP (Dako Ltd, UK).