Pe conjugated anti b220

Flow cytometry analysis of spleen cell suspensions was performed using the following fluorescent-labeled anti-mouse antibodies: PE-conjugated anti-B220 (BD Pharmingen #553089), PE-Cy7-conjugated anti-CD95 (BD Pharmingen #557653), Alexa Fluor 647-conjugated anti-GL7 (BD Pharmingen #561529), APC-conjugated IgG1 (BD Pharmingen #560089), APC-Cy7-conjugated anti-CD3 (Biolegend #100222), and PE-conjugated anti-Gr1 (eBioscience #12-5931-81). DAPI (4',6-diamidino-2-phenylindole) was used for the exclusion of dead cells. Cell cycle analysis of GC B cells was performed at day 10 after NP-CGG immunization, 2 h after bromodeoxyuridine (BrdU) intraperitoneal injection (2 mg), following the manufacturer’s instructions (V450-conjugated anti-BrdU, BD Horizon #560810). Data were acquired on a MACSQuant Flow Cytometer (Miltenyi Biotech) and analyzed using the FlowJo v10.1 software (TreeStar).

Antibodies for lineage negative (Lin⁻) selection (B220, Gr-1, TER-119, CD4 and CD8), for sorting (anti-Sca-1-PE, Anti-c-kit-APC, biotin-conjugated lineage panel antibodies (B220, Gr-1, CD3ε, TER-119) and streptavidin-conjugated APC-Cy-7) and for multilineage engraftment analysis (PE conjugated anti-B220, Thy-1.2, Mac-1 and Gr-1) along with the appropriate isotypes were purchased from BD Pharmingen. Sheep anti-rat IgG Dynabeads® were purchased from Invitrogen. Biotinylated murine anti-CD34 was purchased from eBiosciences. CD105, CD106, CD90, CD39, CD11b were from Biolegend. Murine GFP, CD45, ASMA, Vimentin, DMP-1, Runx-2, periostin, BSP and ALP antibodies were purchased from Abcam. Osteocalcin (OCN) antibody was from Takara Bio (Clontech). DSPP antibody was from Santa Cruz Biotechnology. All secondary antibodies were from Jackson ImmunoResearch Laboratories. CD45-APC for flow cytometry was purchased from eBiosciences.