B220 cd45r fitc

Lymphoma immunophenotype was assessed as previously described [22 (link)]. The following antibodies were used for staining: CD19-APC, B220 (CD45R)-FITC, IgM biotin, IgD-PE, and Streptavidin-APC/Cy7 (BD Biosciences, USA). Data were collected by a flow cytometer (BriCyteE6, Mindray Bio- Medical Electronics Co. Ltd., Shenzhen, China) and analyzed using FlowJo Version 10.1 software. For the analysis of tumor-associated macrophages, cell suspensions of lymph nodes were obtained by grinding and filtering tissues through 0.4-μm cell strainers (BD Biosciences, USA) in PBS. Cells were then transferred to a fresh tube for centrifugation at 1000× g for 5 min. The cell pellet was incubated in red blood cell lysis buffer (Lonza) for 1 min at room temperature, diluted in PBS, and centrifuged for 1000× g for 5 min. The cell pellet was incubated with fluorescent-conjugated antibodies (dilution 1:50 in PBS) for 15 min at 4 °C in the dark, washed, and analyzed by flow cytometry. The following antibodies were used: F4/80 (6F12) from Miltenyi; CD11b (M1/70.15.5) and Gr1 (RB6–8C5) from BD Pharmingen.

The following anti-mouse monoclonal Antibodies (mAbs) were used for flow cytometry: CD25 APC, IgM APC, CD3e Pacific Blue, CD11b Pacific Blue, CD3 FITC, CD8a FITC, IgD FITC, MHC-II FITC, CD39 PE-Cyanin7 (all eBioscience, Waltham, MA, USA); CD4 APC/Cy7, CD21/CD35 APC/Cy7, IgD APC/Cy7, CD73 Pacific Blue, CD8a PE, B220/CD45R PE-Cyanin7, CD19 PE-Cyanin7, F4/80 PE-Cyanin7, Ly6G PerCP/Cy5.5 (all BioLegend, San Diego, CA, USA); CD11c APC, CD45 APC/Cy7, B220/CD45R FITC, CD23 PE, CD44 PE, CD4 PerCP (all BD Biosciences, San Jose, CA, USA); IgM PE (all SouthernBiotech, Birmingham, AL, USA); Ly6C PE, Ter119 PE (all Miltenyi Biotech, Bergisch Gladbach, Germany).