Fitc conjugated cd4

Blood tests, including complete blood count, peripheral neutrophil/lymphocyte ratio (NLR), aspartate aminotransferase, alanine aminotransferase (ALT), total bilirubin (T-Bil), serum albumin, and serum creatinine, were performed in a professional clinical laboratory (Japan Clinical Laboratories, Kyoto, Japan).
To evaluate the immune function, the CD4/CD8 T-cell ratio was analyzed. The conjugated mouse anti-rat monoclonal antibodies used for flow cytometry, APC-conjugated CD3, FITC-conjugated CD4, and PE-conjugated CD8a, were commercially available (BD Biosciences, San Josè, CA, United States). Samples were acquired using a BD Accuri C6 (BD Biosciences).
The fecal IgA content was determined to evaluate intestinal barrier function by enzyme-linked immunosorbent assay (ELISA) using a rat IgA ELISA kit (Bethyl Laboratories, Inc., Montgomery, TX, United States). Serum lipopolysaccharide (LPS) levels were evaluated using a rat LPS ELISA kit (CUSABIO, Wuhan, China).

MLE-12 cells were stained with PE-Annexin V (BD Pharmingen) and 7-amino-actinomycin D (BD Pharmingen). Splenocytes from PBS- and pristane-injected mice were stained for FITC-conjugated CD4 (BD Pharmingen), PE-Annexin V and 7-amino-actinomycin D. These stained cells were analyzed by flow cytometry. Annexin V-positive and 7-amino-actinomycin D-negative cells were defined as apoptotic cells. The average apoptotic percentages of MLE-12 cells without stimulation, or CD4+ splenocytes from PBS-injected mice on day 14, were determined as 1.0 (apoptotic cell ratios).

PBMCs were isolated from RA patients using density-gradient centrifugation on Ficoll-Paque. The cell pellets were washed and resuspended with PBS containing 1% FBS. Then, the cells were incubated with FITC-conjugated CD4, PE-Cy5-conjugated CD3, APC-conjugated CD25 and PE-conjugated CD127(all from BD Pharmingen, USA). For intracellular cytokine staining, the cells were pretreated with Cell Activation Cocktail (Thermo) for 6h. Then, cells were washed and stained with PE-conjugated CD4 and AF750-conjugated CD3 antibodies (all from BD Pharmingen, USA). After fixed and permeabilized with Transcription Factor Buffer Set (BD Pharmingen, USA), the cells were stained with AF700-conjugated IL-17A (Biolegend, USA), FITC-conjugated IFN-γ and PE-Cy7 conjugated IL-4 antibodies (all from BD Pharmingen, USA). For Foxp3 staining, cells were fixed and permeabilized, and then stained with BV421 conjugated Foxp3 antibody (Biolegend, USA). Multiparameter flow cytometry (DxFLEX, Beckmancoulter) and FlowJo software (Tree Star) were adopted for the data analysis of stained cells.
Cytometric bead array (CBA) was used to measure IL-6, IFN-γ, IL-4, IL-17A and IL-10 levels in serum from RA patients or health controls. The measurement was performed using Aimplex Human Th1/Th2/Th17 14-plex cytokine kit (Quanto Bio, China) according to the manufacture’s instruction.

The single‐cell suspension was obtained from the spleen, lymphocyte node (LN), blood and tumour of HNSCC mouse model as previously described 21. The following antimouse antibodies were used for staining: FITC‐conjugated CD4, CD8 and CD11b, PE‐conjugated B7‐H3 and Gr‐1 (all from Becton Dickinson, Mountain View, CA, USA); PerCP‐Cy5.5‐conjugated F4/80, PE‐conjugated IFN‐γ, mouse regulatory T cell staining kit #3 (all from eBioscience, San Diego, CA, USA); and isotype‐matched IgG controls (eBioscience). The cells were analysed using FlowJo (Tree Star, Ashland, OR, USA) and gated by the side scatter and forward scatter filters. Dead cells were excluded by staining 7AAD (Invitrogen, Carlsbad, USA).

The single‐cell suspensions from spleens, draining lymph node (LN), blood, and tumor from WT and Tgfbr1/Pten 2cKO mice were processed according to a standardized protocol (Trellakis et al., 2013). Tumors from Tgfbr1/Pten 2cKO mice were excised and digested and processed using a gentle Macs dissociator and a murine tumor dissociation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Flow cytometry analysis of cells was performed by flowjo (Tree Star, Ashland, OR, USA), and cells were gated by surface markers and negative controls (Yu et al., 2016). Death cells were excluded by staining with 7AAD (Invitrogen). The following anti‐mouse antibodies were used for fluorescence staining: FITC‐conjugated CD4, CD8, and CD11b; PE‐conjugated TIM3 and Gr‐1 (all from Becton Dickinson, Mountain View, CA, USA). Cells stained with isotype‐matched IgG were used as negative controls (eBioscience, San Diego, CA, USA).