Percp cy5.5 conjugated anti cd19

Flow cytometry analysis was performed as described [22 (link)]. The following antibodies were used to stain cells at 4° C for 20 min: anti-ROR1 mAb (4A5) conjugated with Alexa-647 (4A5-Alexa-647) was generated in our laboratory. Anti-CD23 mAb conjugated with FITC (anti-CD23-FITC) was ordered from Life Technologies (Cat #MHCD2301). Anti-CD200 mAb conjugated with PE (anti-CD200-PE) was obtained from BD Biosciences (Cat #552475). PerCP-Cy5.5 conjugated anti-CD19 was purchased from Ebioscience (Cat #45-0199-42) and PE conjugated anti-CD5 antibodies were from BD Biosciences (Cat, #555355). The stained cells were washed twice with FACS buffer (phosphate buffered saline, pH 7.4 (PBS), 3% FBS), and examined by four-color, multiparameter flow cytometry using a dual-laser FACSCalibur (BD Biosciences), and the data were analyzed using FlowJo software (TreeStar). We subtracted the mean-fluorescence intensity (MFI) of cells stained with a fluorochrome-labeled, isotype-control mAb from the MFI of the same cells stained with each antibody to determine the specific increase in MFI (ΔMFI).

For detection of Tfh cells, human PBMCs were stained with Alexa Fluor 647-conjugated anti-CD4, Alexa Fluor 488-conjugated anti-CXCR5, and phycoerythrin (PE)-conjugated anti-PD-1 (all from BD Pharmingen, San Jose, CA). Cells were gated for CD4⁺ T cells first and then for CXCR5⁺PD-1⁺ Tfh cells. For detection of Breg cells, PBMCs were stained with PerCP/Cy5.5-conjugated anti-CD19, fluorescein isothiocyanate (FITC)-conjugated anti-CD5, and PE-conjugated anti-CD1d (eBioscience) for 15 minutes. CD5⁺CD1d^high cells were analyzed with a CD19⁺ gate.
For intracellular IL-10 staining, PBMCs were incubated for 24 hours with 10 µg/ml LPS and stimulated with PIB for the last 5 hours. Surface staining with PerCP/Cy5.5-conjugated CD19 or FITC-conjugated anti-CD5 was first performed for 15 min, and cells were re-suspended in Fixation/Permeabilization solution (Invitrogen). Intracellular staining of PE-conjugated anti-IL-10 was performed according to the manufacturer’s protocol (eBioscience). After staining, IL-10⁺ cells were analyzed with a CD19⁺ gate by flow cytometry. For some experiments, cells were stained with FITC-conjugated CD19 and PE-conjugated anti-IL-10 (eBioscience) and detected by immunofluorescence microscopy.