Igm biotin

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.

Mononuclear cells were immunostained with various combinations of the following fluorescence-conjugated antibodies: CD4 (eBioscience, San Diego, CA, USA), CD25 (eBioscience), Foxp3 (eBioscience), IFN-γ (eBioscience), interleukin (IL)-4 (BD Pharmingen, San Diego, CA, USA), IL-17 (eBioscience), B220 (BioLegend, San Diego, CA, USA), IgD (eBioscience), IgM biotin (BD Pharmingen), CD138 (BD Pharmingen), CD23 (eBioscience), CD21 Biotin (eBioscience), Fas (CD95) (BD Pharmingen), and streptavidin (BD Pharmingen). Before intracellular cytokine staining, the cells were stimulated in a culture medium containing phorbol myristate acetate (25 ng/ml; Sigma-Aldrich, St. Louis, MO, USA), ionomycin (250 ng/ml; Sigma-Aldrich), or monensin (GolgiStop, 1 μl/ml; BD Pharmingen) in an incubator under an atmosphere of 5% CO₂ at 37°C for 4 h. Intracellular staining was performed using an intracellular staining kit (eBioscience) according to the manufacturer’s protocol. Flow cytometric analysis was performed on a FACS LSRFortessa instrument (BD Biosciences, Franklin Lakes, NJ, USA).

The following antibodies comprising the B cell antibody panel were used: B220-V500, CD19-PerCP Cy5.5, CD23-PE, CD21-APC, CD24-PECy7, CD86-V450, MHCII-FITC, and IgM-Biotin (BD Bioscience, Erembodegem, Belgium). T cells panel consisted of the following antibodies: CD4-PerCP, CD3-AlexaFluor 700, CD62L-V500, CD44-FITC, CD28-PE, and CXCR5-V450 (BD Bioscience, Erembodegem, Belgium). Cells were acquired on a FACS Fortessa machine (BD Immunocytometry system, San Jose, CA, USA) and data was analyzed using Flowjo software (Treestar, Ashland, OR, USA).