Fitc conjugated anti cd19

Peripheral blood mononuclear cells from APDS1 (four patients), APDS2 (four patients), APDS-L (four patients), CVID (four patients), HIGM (one patient), and 24 adult healthy controls were subjected to flow-cytometry analysis. We assessed pAKT at Ser473 by flow cytometry as follows. PBMCs were suspended at a density of 1 × 10⁶ cells/μL in serum-free RPMI with or without 10 µM of 110δ inhibitor (IC87114) in the presence of FITC-conjugated anti-CD19 (HIB19) (BD Biosciences). The cells were incubated for 20 min at 37°C and washed twice. They were fixed and permeabilized according to the BD Phosflow protocol (protocol III). They were then stained and subjected to flow cytometry. The following antibodies were used for staining: Alexa Fluor 647-conjugated anti-phospho AKT (Ser473) (D9E) (Cell Signaling Technology), FITC-conjugated anti-CD19 (BD Biosciences), PE-conjugated anti-CD3 (UCHT1) (BD Biosciences), PE-conjugated anti-CD14 (Mφ97) (BD Biosciences), or FITC-conjugated anti-CD56 (C5.9) (SIGMA-ALDRICH), PE-conjugated CD16 (3G8) (BD Biosciences), and PerCP-Cy 5.5-conjugated anti-CD10 (HI10a) (BD Biosciences). Negative selection of B cells from PBMCs was performed using Pan B-Cell Isolation Kit, human (Miltenyi Biotec Inc., Auburn, CA, USA).

Fresh CD34⁺ cells from human umbilical cord blood were cultured in expansion culture medium for 7 days in the presence of TNFSF15 at 37°C with 5% CO₂. Then cells were collected, respectively, washed and stained with PE.Cy7‐conjugated anti‐CD3 (BD; 557851), APC.Cy7‐conjugated anti‐CD33 (BioLegend; 366613), FITC‐conjugated anti‐CD19, PerCP‐conjugated anti‐CD56 (BD; 560842) and APC‐conjugated anti‐CD235a (BD; 561775) for 30 minutes under room temperature in dark. Then the cells were resuspended and analysed by flow cytometry.

The following anti-human monoclonal antibodies (mAbs) were used for surface markers and intracellular IL-10 detection: fluorescein isothiocyanate (FITC)-conjugated anti-CD19; phycoerythrin (PE)-conjugated anti-CD24 (BD Biosciences, San Diego, USA); PE-Cyanine7-congugated anti-CD38; allophycocyanin (APC)-conjugated anti-IL-10 (Biolegend, San Diego, USA); and isotype-matched and fluorochrome-matched control antibodies. Cells were stained with combinations of CD19-FITC, CD24-PE and CD38-PE-Cy7 mAbs for surface phenotype. For intracellular IL-10 detection, cultured cells were washed, fixed, permeabilized, and stained with IL-10-APC mAb. APC-conjugated isotype control was used for gate setting for cytokine expression. Stained cells were analyzed on an eight-color FACSCanto II flow cytometer (BD Biosciences) using FACSDiva software (BD Biosciences).