Percp conjugated cd19

Evaluation of Breg cells frequencies by flow cytometry, circulating Breg cells were detected using FITC-conjugated-CD38, PE-conjugated-CD24 (BD Bioscience, USA), and PerCP-conjugated CD19 (BD Bioscience, USA).100 μl of blood sample was incubated with 10 μl of CD24, CD38, and CD19 for 20 minutes at 4 C in the dark. Following incubation, red blood cell lysis, washing, and analysis by FACS Calibur flow cytometry with Cell Quest software (Becton Dickinson Biosciences, USA) was done. An isotype-matched negative control was used for each sample. A Forward and side scatter histogram was used to define the lymphocytes. Then CD19+ B cells were gated. Then the expression of CD38 and CD24 on the CD19+B cells was revealed. Regulatory B cells were specified as CD19⁺CD24^highCD38^high cells (Figure 1).

Using flow cytometry, circulating Bregs were detected using FITC-conjugated-CD38, PE-conjugated-CD24 (Bioscience, USA), and PerCP-conjugated CD19 (BD Bioscience, USA). Briefly, 100 µl of blood sample was incubated with 10 µL of CD24, CD38 and CD19 for 20 min at 4 °C in the dark. Following incubation, RBCs were lysed and washed. Cells were fixed and permeabilized then stained with APC-conjugated IL-10 (BD Bioscience, San Jose, CA, USA) and analysis by FACS Calibur flow cytometry with CellQuest software (Becton Dickinson Biosciences, San Jose, CA, USA). An isotype-matched negative control was used for each sample. Forward and side scatter histograms were used to define the lymphocytes population. CD19⁺ IL-10⁺ B cells were gated, then the expression of CD38 and CD24 on the CD19⁺B cells were detected. Bregs were identified as CD19⁺ IL-10⁺CD24^+hiCD38^+hi cells. CD19⁺ B cells were selected based on the use of the isotype-matched negative control. However, for proper gating of the IL-10⁺ and CD24^+hiCD38^+hi cells, fluorescence minus one controls were employed, shown in light blue and red colors in the dot blots for the gated cells and controls, respectively (Figure 1A).

Circulating B lymphocytes and Bregs were detected in peripheral blood using the following markers (BD Biosciences, San Jose, CA, USA): PerCP-conjugated CD19, FITC-conjugated CD24 and PE-conjugated CD38. Briefly, the blood sample (100 μl) was incubated with 10 μL of the markers (CD19,CD24 and CD38) for 20 min at 4°C in the dark. RBCs were lysed using BD FACS Lysing solution (BD Biosciences, San Jose, CA, USA), and washing was done after incubation. Gating of CD19⁺ B cells was done, followed by CD38⁺ and CD24⁺ expression on CD19⁺ B cells. The analysis was done as described previously. CD19⁺CD24^+hiCD38^+hi cells were identified as Bregs. (Fig 3)