Anti b220 fitc

Cells were incubated on ice for 45 min in Fc block in the presence of relevant primary antibodies. The anti-CD11c FITC, anti-CD11b PE, anti-CD103 PECy5, anti-MHCII APC-efluor780, anti-F4/80 efluor450, anti-CD45 BV510, anti-CX3CR1 PECy7, anti-Sca1 PE, anti-CD16/32 PerCP, anti-CD34 efluor450, anti-B220 FITC, anti-Gr1 FITC, anti-Ter119 FITC, anti-CD3 FITC, and anti-CD127 APC were purchased from eBioscience. The anti-CD11b PE, anti-CD11c APC, anti-Ly6C FITC, anti-CD117 PECy7 and anti-CD43 PerCP were purchased from BD Bioscience. After staining, cells were analyzed by flow cytometry on a FACS Canto. Data were analyzed with the FlowJo software.

Colons were cut into ~1 cm² pieces, washed in 1X Hank’s Balanced Salt Solution (HBSS) containing 2% heat-inactivated fetal bovine serum (FBS), disrupted by vortexing, further treated with collagenase IV (20 mg/ml) and DNase I (10 mg/mL) in RPMI medium and passed through a 70 μM cell strainer to collect lamina propria cells. Cells were treated with blocking antibodies to CD16/32 (1:100), and stained for viability using Zombie Viability Dye V500 (1:400). Surface staining was done on ice for 20 min using anti-CD45 APC-eFluor780 (1:400, clone 30F11, eBioscience), anti-CD11b APC (1:400, clone M1/70, InVitrogen), anti-Gr1 PerCp-Cy5.5 (1:300, clone RB6-8C5, Biolegend), anti-Siglec F PE (1:400, clone E50-2440, BD), anti-CD3 FITC (1:200, clone 145-2C11, Biolegend), anti-CD49b FITC (1:200, clone DX5, eBioscience), anti-B220 FITC (1:300, clone RA3-6B2, eBioscience), anti-CD4 PerCp-Cy5.5 (1:400, clone RM4-5, eBioscience), and anti-CD8a AlexaFluor 700 (1:200, clone 53–6.7, Biolegend),. Intestinal epithelial cells were collected as described in^{22 (link)}. Cells were stained with anti-EpCAM PE-Cy7 (1:400, clone G8.8, eBioscience) and anti-FITC CD44 (1:200, clone IM7, eBioscience). Cells were analyzed on the Fortessa (BD Biosciences) and data analysis was performed on FlowJo (Tree Star).

For leukocyte surface marker determination, pooled LNCs or splenocytes were obtained from mice (as described in the mouse lymphocyte proliferation assay). Cell suspensions were prepared as described previously [31 (link)]. For immune phenotyping, the following antibodies were used: anti-CD4 FITC, anti-CD8 FITC, anti-CCR5 PE, anti-B220 FITC, anti-CD11c PE, anti-MHC class II FITC, and anti-CD11b PE (all from eBioscience, San Diego, CA, USA). Stained cells were counted with a FACS Calibur flow cytometry kit (FC500, Beckman Coulter).

Mouse kidneys were fixed in 10% phosphate-buffered formalin, embedded in paraffin, sectioned at 5 μm thickness, and stained with H&E, as described (41 (link)). Glomerular size was measured using ImageJ software. Glomerular area was averaged from about 50 glomeruli from 3 independent sections. Spleens were cryo-sectioned and processed by standard fluorescence immunohistochemical methods, as described (41 (link)). Sections were stained with appropriate combinations of anti-B220-FITC, anti-B220-allophycocyanin, anti-CD4-FITC (all from eBioscience), anti-Gr-1-biotin (BD Biosciences), and streptavidin-Cy3 (Invitrogen). Fluorescence images were acquired using an LSM510 confocal microscope (Zeiss). To observe nuclear morphology, sorted SDMCs were smeared on slides by cytospin centrifugation, air-dried and stained with Diff-Quik solution (Sysmex Corporation). Images were acquired on an Olympus BX51 microscope equipped with an Olympus DP70 digital camera.