Anti cd4 alexa fluor 700

CD4, CD8, and CD56 positive cells in PBMCs isolated from healthy human donors were detected by flow cytometry on a fluorescence-activated cell sorter FACScan (Becton Dickinson, Walpole, MA, USA). Alexa Fluor 700 anti-CD4 (eBioscience, San Diego, CA, USA), anti-CD8-Per-CP-Cy5.5, and anti-CD56-Per-CP-Cy5.5 (BD Pharmingen, San Diego, CA, USA) antibodies were used to detect expression levels of CD4, CD8, and CD56, respectively. Approximately 1 × 10^⁶ pelleted PBMC cells were blocked in PBS buffer with 1% BSA for 20 min at room temperature. The cells were then stained with antibodies at 4 °C for 30 min, washed twice in PBS buffer with 1% BSA and resuspended in 0.5 ml staining buffer for FACScan analysis.

For Th17 cell immunostaining, purified CD4⁺ T-cells were identified using labeled monoclonal antibodies against the following molecules: Alexa Fluor 700 anti-CD4 and PE anti-ROR-γt (AFKJS-9 clone, eBioscience, San Diego, CA, USA).
For Treg immunostaining, purified CD4⁺ T-cells were identified using labeled monoclonal antibodies against the following molecules: Alexa Fluor 700 anti-CD4 (RPA-T4 clone, BD Pharmingen), PE-Cy5 anti-CD25 (M-A251 clone, BD Pharmingen), Alexa Fluor 647 anti-CD127 (HCD127 clone, BioLegend), and Alexa Fluor 488 anti-FOXP3 (150D clone, BioLegend).
To analyze CTLA-4 expression in Treg cells, PE anti-CTLA-4 antibody (BNI3 clone, BD Pharmingen) was added. The integrated mean fluorescence intensity (iMFI) was calculated by multiplying the percentage of CTLA-4 by their corresponding mean fluorescence intensity (MFI), as described previously [26 (link)].
Intracellular staining for FOXP3 and ROR-γt CD4⁺ T-cells was performed using a commercially available kit (BioLegend) following the manufacturer's instructions. Appropriate isotype controls were used to allow identification of positive and negative cell populations. To rule out nonspecific antibody binding and autofluorescence, quadrants were set according to isotype controls. Positive and negative gates for each molecule were also verified on the basis of “fluorescence-minus-one” controls.

Single cell suspension of colon cells were isolated as previously described [41 (link), 42 (link)] and analyzed by flow cytometry. Cells were stained with the following antibody panels: panel 1) CD45, CD11b, CD11c, F4/80, MHC II, Ly-6C, and Ly-6G; panel 2) CD45, CD11b, CD3, CD4, CD8, CD19, and MHC II. Flow cytometry was performed with the FACS LSR Fortessa (BD Biosciences) and data were analyzed using DIVA software (BD Biosciences). The following antibodies were used: anti-F4/80 BV421 (cat. 565,411), anti-Ly-6C Alexa Fluor 700 (cat. 561,237), anti-CD11c PE (cat. 561,044), anti-MHC II BV650 (cat. 563,415), anti-CD3 BV510 (cat. 563,024) from BD Bioscences (Milano, Italy); anti-CD11b PerCP-Cy5.5 (cat. 45–0112), anti-CD4 Alexa Fluor 700 (cat. 56–0041), anti-Ly-6G FITC (cat. 11–5931), anti-CD45 Pe-Cy5 (cat. 15–0451), anti-CD8 PE (cat. 12–0083), anti-CD19 FITC (cat. 11–0193) from EBiosciences (San Diego, CA, USA).