Fitc conjugated anti cd4

Cells from the BALF were stained with surface markers, including PE-Cy7 conjugated anti-CD45 (BD Biosciences, CA, USA), AF488 conjugated anti-CD11b (BD Biosciences, CA, USA), and PE conjugated anti-Ly6G (BD Biosciences, CA, USA). Cells from blood and spleen were stained with various surface markers, including PE-Cy7 conjugated anti-CD45 (BD Biosciences, CA, USA), Percp-cy5.5 conjugated anti-CD3 (BD Biosciences, CA, USA), FITC conjugated anti-CD4 (BD Biosciences, CA, USA), PE conjugated anti-CD25 (BD Biosciences, CA, USA), and APC conjugated anti-CD39 (BD Biosciences, CA, USA).
Peripheral blood mononuclear cells (PBMCs) from ARDS patients and healthy donors were stained with PerCP conjugated anti-CD3 (BD Biosciences, CA, USA), FITC conjugated anti-CD4 (BD Biosciences, CA, USA), APC conjugated anti-Foxp3 (BD Biosciences, CA, USA), and PE conjugated anti-CD39 (BD Biosciences, CA, USA). This study was approved by the Jinling Hospital Ethics Review Committee and written informed consent was provided by all subjects or their legal representatives.

50 μL of peripheral blood from healthy controls and pSS patients were added into the flow cytometry tube. The cells were then stained with 5 μL fluorescence-conjugated Abs: PerCP-Cy5.5-conjugated anti-CD3 Ab (BD Biosciences, USA), FITC-conjugated anti-CD4 (BD Biosciences, USA), and APC-conjugated anti-CD8 (BD Biosciences, USA). After mixing, the cells were incubated at room temperature for 15 minutes in the dark, and 1 mL of red blood cell lysis solution was added. After lysis at room temperature for 5 minutes, 3 mL of staining buffer (PBS containing 2% FBS) was added into the cells, followed by washing twice at 1500 rpm for 5 minutes. Flow cytometry was then used for detection. BD Cellquest software was used to obtain data by circling out CD3⁺ cells on the forward scattered light (FSC) and side scattered light (SSC) dot plots. Proportion of CD4/CD8 double-positive T cells to CD3⁺ cells was further analyzed. Flow data analysis and graphing used Flowjo software (Tree Star, USA).

Spleen cells were prepared as described above. The cells were surface-stained with fluoresce in isothiocyan (FITC)-conjugated anti-CD4 (BD Pharmingen, San Diego, CA, USA) and PE-conjugated anti-CD25 (eBioscience) to separate T cells. For analysis of the FoxP3, cells were fixed, permeabilized and stained according to the manufacture instruction for FoxP3 staining (PE-Cy5-conjugated anti-mouse/rat FoxP3 staining kit; eBioscience). To analyze intracelluar cytokine production, the cells were cultured for 48 h in the prescence of 2 mM monensin (BD Pharmingen) with 100 ng/ml phorbolmyristate acetate (PMA) (Alexis, Lausen, Switzerland) and 1 mM ionomycin (Alexis). After washed and blocked with Fc-blockade (CD16/32; BD Pharmingen) for 30 min, cells were directly surface-stained with PE-Cy5-conjugated anti-CD3e (BD Pharmingen) and FITC-conjugated anti-CD4. After fixed and permeabilized using the BD cytofix/cytoperm kit (BD Pharmingen), the cells were stained using PE-conjugated anti-IL-4 (BD Pharmingen) or PE-conjugated anti-IFN-γ (BD Pharmingen). Cells were analyzed (10⁴ gated events were collected) using a FACSCalibur. Background fluorochrome was assessed using appropriate isotype and fluorochrome-conjugated control mAbs. Data collected were analyzed using FlowJo software.