Flow Cytometry Analysis of Immune Cells

The number of B cells (B220⁺) and T cells (CD3⁺), the proportion CD4⁺ T cells, dead B and T cells and the proportion of PNA^hi B cells, plasma cells (B220^lo CD138⁺) and NP-binding CD38^hi IgG1⁺memory B cells were determined by FACS analysis using a FACSCalibur ™ flow cytometer (BD Biosciences). Single cell suspensions were prepared from spleens or Peyer’s patches of Hoxc4^−/− and Hoxc4^+/+ mice and stained with Phycoerythrin (PE)-labeled anti-mouse B220 mAb (clone RA3-6B2) (eBioscience Corp.), fluorescein isothiocyanate (FITC)-labeled anti-mouse CD3 mAb (clone 17A2) (BioLegend, Inc.), PerCP-anti-mouse CD4 mAb (clone GK1.5) (BioLegend, Inc.), 7-AAD (BD Biosciences), FITC-PNA, FITC-anti-mouse CD138 mAb (clone 281-2) or APC-anti-mouse IgG1 (clone X56) (BD Biosciences), PE-NP (Biosearch Technologies, Inc.) and PECy7-anti-mouse CD38 mAb (clone 90) (eBiosciences Corp.). Single B220⁺ cell suspensions were prepared from spleens or Peyer’s patches using the EasySep® Mouse B Cell Enrichment Kit (StemCell Technologies Inc.). For the preparation of PNA^hi (GC) B cells, spleen or Peyer's patches B cells were stained with PE-anti-mouse CD45R (B220) mAb (clone RA3-6B2) (BD Biosciences) and FITC-PNA. Labeled lymphocytes were then sorted using a MoFlow™ cell sorter (Dako), yielding 95% pure PNA^hi B220⁺ cells.