Pacblue conjugated anti cd45 clone 30 f11

The small intestine and/or colon were removed and flushed with ice-cold sterile PBS using a 19-gauge feeding needle. The small intestine was further subdivided into the duodenum (proximal 7 cm), jejunum (next 10 cm), and ileum (distal 10 cm), and Peyer patches were removed. Both small intestinal regions and colons were then opened longitudinally and gently agitated at 4°C in PBS, 2% FBS, 5 mM HEPES, and 1 mM DTT for 10 min. The tissue was then transferred into prewarmed PBS, 2% FBS, 5 mM HEPES, and 5 mM EDTA and rotated at 37°C for 15 min followed by vigorous shaking to remove epithelial cells. This was repeated and epithelial cells from both fractions were combined and washed with PBS. The epithelium was then digested in DMEM containing 10% FBS, 0.5 U/ml Dispase II (StemCell Technologies), and 50 μg/ml DNase (Roche) for 10 min at 37°C. The resulting solution was passed through 40-μm filters and washed with PBS, 2% FBS, and 1 mM EDTA. The resulting single-cell suspension was initially Fc blocked with anti-CD16/CD32 (clone 93; BioLegend) and then stained with the following Abs: PacBlue-conjugated anti-CD45 (clone 30-F11; BioLegend) and allophycocyanin-conjugated anti-EpCam (clone G8.8; BioLegend). Cell viability was assessed by propidium iodide (BioLegend) staining.