Alexa fluor 647 conjugated anti rat antibody

MSC cultures were fixed with 4% paraformaldehyde for 10 min, and blocked in PBS buffer containing 5% BSA (Sigma-Aldrich) for 60 min at room temperature. After blocking, cells were incubated with primary antibody in DPBS buffer containing 1.2% BSA overnight at 4°C. As primary antibodies, 1:200 anti-PD-1 (clone: RMO1-14, Biolegend,) and 1:200 anti-MLANA (Byorbit) were used. After incubation, cells were washed three times in PBS, and incubated for 1 h at room temperature with 1:100 Alexa Fluor 647-conjugated anti-rat antibody (Jackson ImmunoResearch Laboratories, Baltimore, PA) or 1:500 Alexa Fluor 555-conjugated anti-rabbit antibody (Thermo Fisher Scientific). Tubulin was stained with 1:500 Alexa Fluor 488-conjugated anti-tubulin-α antibody (clone: 10D8, Biolegend). Nucleus staining was performed using DAPI (Life Technologies). Slides were washed three times with DPBS between each step. Images were obtained at 60x magnification using an Olympus confocal laser scanning microscope.

Cells from spleen or peritoneal flushes were stained with one or more of the following anti-mouse MAbs: CD3-fluorescein isothiocyanate (FITC), CD3-phycoerythrin (PE)/Cy7 (145-2C11), GL7-eFluor 450 (GL7), F4/80-FITC (BM8), CD4-eFluor 450 (RM4-5), CD44-peridinin chlorophyll protein (PerCP)-Cy5.5 (IM7), PD-1–PE (J43), IgM-PE-Cy7 (11/41), and CD5-PerCP-Cy5.5 (53-7.3) from EBioscience; B22 0PE (RA3-6B2), CD95-PE-Cy7 (Jo2), CD19-allophycocyanin (APC)-Cy7 or -APC (ID3), CD21-FITC (7G6), and CD23-PE (B3B4) from BD Pharmingen; B220BV510 (RA3-6B2) and CD138-PE (281-2) from BioLegend; and IgG2b-FITC and IgG2c-FITC (polyclonal) from Southern Biotech. Rat anti-mouse CXCR5 (551961) (BD Pharmingen) was detected with an Alexa Fluor 647-conjugated anti-rat antibody (712-606-153) from Jackson ImmunoResearch, Inc. Cells were then assessed on a CyAn ADP flow cytometer and analyzed with FlowJo 9.3.2. (TreeStar). For intracellular staining, cells were first stained for surface markers and then fixed and permeabilized with Cytofix/Cytoperm Plus (BD Biosciences).