Igg2b

Single-cell suspensions were labeled with fluorescently conjugated anti-mouse antibodies, including anti-B220, -CD21, -CD23, -CD38, -CD86, -CD95 (Fas), -CD138 (Syndecan), -GL7, -CXCR4, -IgM, -IgD, and -IgG2b (BD Biosciences, eBiosciences, Biolegend). A LIVE/DEAD Fixable Dead Cell Stain Kit (Molecular Probes) was used to identify viable cells. Different spleen B cell populations were defined as following: T1 (B220⁺CD23⁻IgM⁺CD21^−/lo), T2-MZP (B220⁺CD23⁺IgM^+/hiCD21^+/hi), FoB (B220⁺CD23⁺IgM^+/loCD21^+/lo), marginal zone B cells (MZB) (B220⁺CD23⁻IgM⁺CD21^hi), germinal center (GC) B cells (B220⁺IgD⁻GL7⁺CD95⁺), GC light zone (LZ) and dark zone (DZ) B cells (B220⁺IgD⁻GL7⁺CD95⁺CD86^+/−CXCR4^+/−), and PC (CD38⁻B220⁻CD138⁺). Fluorescence minus one controls were used when defining different populations of B cells and isotype controls were used in switching experiments. For Ig class switching, single-cell suspensions were fixed with formaldehyde, permeabilized with saponin and labeled with biotinylated antibodies to IgM, IgG1, IgG2b, IgG3, IgE, IgA, or isotype controls (BD Biosciences). Streptavidin-FITC was used as a second step. Data were acquired on FACSCalibur, FACSVerse, or LSR Fortessa (Becton Dickenson). Analyses were made using FlowJo (TreeStar, Inc.).

Target cells were adjusted to a density of 1.0 × 10⁶/mL, and 100 μL of cell suspension was transferred into flow tubes. The primary antibodies used were as follows: mouse anti-rat CD11b (BD, New Jersey, USA) and CD68 (Santa Cruz, Dallas, Texas, USA), and mouse IgA (BD) and IgG2b (BD), and were used for the identification of M0 macrophages; mouse anti-rat CD29 (eBioscience, San Diego, California, USA), CD90 (eBioscience), CD44 (Santa Cruz), CD79a (Santa Cruz), CD45 (eBioscience), and CD11b (BD) were used for the identification of PBMSCs; and mouse anti-rat CD206 (Santa Cruz) and CD86 (Santa Cruz), and mouse IgG1 (BD) and IgG2b (BD) were used for the identification of M1 and M2 macrophages. After 30 min of incubation with primary antibodies, cells were subsequently incubated with Alexa Fluor-conjugated rabbit or goat anti-mouse IgG secondary antibodies. After washing with PBS and fixing with 200 μL 4% paraformaldehyde, cell samples were analyzed by FACSCalibur flow cytometry (Becton Dickinson, USA), and data were analyzed using Cell Quest software.

cMSCs and pbMSCs were defined according to the 3 criteria of the International Society for Cellular Therapy [13 (link)]: (1) adhesion to plastic, (2) expression of a specific combination of surface markers, and (3) differentiation potential (trilineage differentiation into adipocytes, osteoblasts, chondrocytes, and blood vascular cells) [10 (link)]. To define the phenotype of cMSCs and pbMSCs, we analyzed cultured cells at passage 4 for rat MSC markers by flow cytometry using the following fluorescence antibodies: SH2, SH3, CD90, CD147, CD34, CD45, and CD133. Mouse IgG1, IgG2a, and IgG2b (Becton Dickinson) were used as isotype controls, and marker expression was evaluated using FACS.