Anti cd45 apc efluor 780

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).

Antibodies used for flow cytometry, immunohistochemisty and western blotting are listed in Table 1. Microglial activation and immune cell influx into the hypothalamus and prefrontal cortex were characterized as previously described (48 (link)). Tissues from the hypothalamus and cortex from each mouse was processed separately as part of a 5-mouse cohort per group, with each experiment repeated 3 times. In brief, mice were perfused with ice cold PBS, brains rapidly removed and hypothalami and prefrontal cortex cell suspensions were generated by mechanical dissociation and applied to a discontinuous 1.03/1.088 percoll gradient. Cells were collected from the interface, blocked with anti-CD16/CD32 (1:300, 553141, BD Biosciences, San Jose, CA), and incubated with anti-CD45 APC-eFluor® 780 (1:300, 47-0451, eBioscience, San Diego, CA) and anti-CD11b PerCP-Cyanin5.5 (1:300, 45-0112, eBioscience, San Diego, CA) in PBS, 5% EDTA, 0.4% BSA. Sytox™ Green dead stain (30 nM, S-34860, ThermoFisher, Chino, CA) was used to exclude dead cells and flow analysis performed using BD LSR II Flow Cytometer. Results were analyzed using FlowJo software (Tree Star, Inc.) and statistical differences were determined by Student's T-test and Tukey's posthoc test.