Fitc conjugated anti cd4 antibody

The blood samples (approximately 300 μL) were collected from mouse eyeball using a heparin sodium anticoagulant tube. The triple volume of red blood cell lysis buffer (Solarbio, Beijing, China) was added into the blood for 10 min and then centrifuged at 400 × g for 15 min. The supernatant was discarded, and the sedimentary leukomonocytes were resuspended with 300 μL PBS twice. Subsequently, 1 μL FITC-conjugated anti-CD4 antibody (BD, US) and 2 μL PE-conjugated anti-CD8 antibody (BD, US) were incubated with leukomonocytes for 30 min in a dark box. Then, leukomonocytes were rinsed with PBS 3 times. A total of 10,000 cells were counted for calculating the percentages of CD4 and CD8 positive cells using flow cytometry (CytoFLEX S, Beckman Coulter, US).

Monocytes were obtained from spleens and mesenteric lymph nodes (MLNs) and subjected to intracellular staining to measure the proportions of CD4⁺IL-17A⁺T cells and CD4⁺CD25⁺FOXP3⁺T cells [29 (link)]. For the analysis of Th17 cells, the cells were treated with a leukocyte activation cocktail with BD GolgiPlug™ (BD Biosciences, New York, U.S.A.) under an atmosphere of 5% carbon dioxide at 37°C for 6 h. The cultured cells were then stained for surface markers with a FITC-conjugated anti-CD4 antibody (BD Biosciences, New York, U.S.A.) and incubated for 15 min at RT in the dark. The cells were next fixed and permeabilized for 20 min at RT in the dark, followed by staining with a PE-conjugated anti-interleukin-17A antibody (BD Biosciences). For Treg cell analysis, the cells were stained with FITC-conjugated anti-CD4 and PerCP-Cy5.5-conjugated anti-CD25 antibodies (BD Biosciences) for 30 min at 4°C in the dark. The cells were then fixed and permeabilized with fixation/permeabilization working solution and permeabilization buffer for 30 min in the dark and subsequently stained with an AF647-conjugated anti-FOXP3 antibody in the dark. The stained cells were washed with permeabilization buffer (BD Biosciences), followed by analysis via flow cytometry.

To determine the concentration of the cytokines including IL-2, IFN-γ, IL-4, IL-6, TNF, IL-17A and IL-10 in the colons and serum of the animals, the BD™ Cytometric Bead Array (CBA) Mouse Th1/Th2/Th17 Cytokine Kit was used according to the manufacturer's instruction. Data were analyzed with FCAP Array software.
For flow cytometry assay, mesenteric lymph nodes (MLNs) and spleen single-cell suspensions were harvested by gently pushing cells through a 70-μm cell strainer with a syringe piston as previously described [34 (link)]. For Treg analysis, cells were stained with FITC conjugated anti-CD4 antibody at 4 ​°C in the dark for 20 ​min. They were fixed and permeabilized for 40 ​min in the dark, and finally stained with PE conjugated anti-FOXP3 antibody. For Th1/Th2/Th17 analysis, cells were first stimulated with a leukocyte activation cocktail in 5% CO₂ at 37 ​°C for 6 ​h. Subsequently, they were stained with FITC conjugated anti-CD4 antibody (BD Biosciences) for 30 ​min in the dark, and then fixed and permeabilized for 40 ​min. Finally, they were stained with APC conjugated anti–IFN–γ antibody (BD Biosciences), BV605 conjugated anti-IL-17A antibody (BD Biosciences), and PE conjugated anti-IL-4 antibody (BD Biosciences). After washing with permeabilization buffer (BD Biosciences), the stained cells were analyzed by flow cytometer.