Cell surface antigen expression on isolated PBMCs was determined by single-parameter fluorescence-activated cell sorter (FACS) analysis with the following monoclonal antibodies (mAbs): (i) FITC-conjugated anti-CD45R/B220, PE-conjugated anti-CCR6, anti-CCR7, anti-CXCR4, anti-CXC5 and PE-conjugated isotype-matched control mAbs (all purchased from R&D Systems, France) and (ii) PE-conjugated anti-CD45R/B220 (BD Biosciences), Alexa Fluor 488 phalloidin (Sigma-Aldrich) and carboxyfluorescein succinimidyl ester (CFSE, Invitrogen). A FACSCalibur flow cytometer (BD-Pharmingen) was used for data acquisition and analysis. After gates were set to include only viable cells, 104 events per sample were collected and analyzed. For each marker, the threshold of positivity was defined relative to the nonspecific binding observed in the presence of the appropriate isotype control mAb.
Pe conjugated anti cd45r b220
Manufactured by BD
Sourced in France
PE-conjugated anti-CD45R/B220 is a monoclonal antibody that targets the CD45R/B220 antigen expressed on B cells. The antibody is conjugated with the fluorescent dye phycoerythrin (PE), allowing for detection and analysis of B cells in flow cytometry applications.
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2 protocols using pe conjugated anti cd45r b220
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Phenotypic Analysis of PBMCs
2
Multicolor Flow Cytometry of Immune Cells
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The following antibodies were used: FITC-conjugated anti-CD11b (cat. no. 557686; clone M1/70; BD Biosciences, Franklin Lakes, NJ, USA), allophycocyanin-conjugated anti-Ly-6B.2 (cat. no. NBP2-13077APC; clone 7/4; Bio-Rad Laboratories, Inc.), FITC-conjugated anti-CD4 (cat. no. MCA2649; clone RM 4–5; eBioscience; Thermo Fisher Scientific, Inc.), PercP-conjugated anti-CD8a (cat. no. 555369; clone, 53–6.7; BD Biosciences), PE-conjugated anti-CD45R/B220 (cat. no. A15835; clone RA3-6B2; eBioscience; Thermo Fisher Scientific, Inc.) for 12 h at 4°C after blocking with 1% bovine serum albumin for 2 h at 37°C. All antibodies were used at a dilution of 1:100. B-lymphocytes were identified as CD11bhiLy6G-7/4hi/lo and macrophagocytes were identified as CD11chi. Cells were washed three times with 0.1% tris-buffered saline-Tween-20. Cells were analyzed using a flow cytometer (LSR II; BD Biosciences). Data was analyzed using BD FACSDiva™ software version 8.0.1 (BD Biosciences).
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