Alexa fluor 594 anti vimentin

Mouse colons were washed in cold CMF-HBSS and placed in CMF-HBSS containing 5 mM EDTA and shaken gently at 37 °C for 30 min to remove epithelial cells. The remaining colon tissue was minced and agitated in RMPI with 2 mg/mL dispase (Gibco), 0.5 mg/mL collagenase I (Gibco) and 0.2 mg/mL DNase (Roche) at 37 °C for 45 min. Tissue was broken up using a 19-gauge needle and filtered through a 100 μM filter. Cells were centrifuged at 350 × g for 20 min and used for flow cytometry. For cell sorting, cells were resuspended in 40%Percoll/RPMI and spun at room temperature for 20 min at 600 × g and the cell pellet was collected. Single-cell suspensions were washed with FACS buffer (PBS/1% FCS) and incubated with combinations of the following Abs: APC-Cy7 anti-CD45 (Clone 30-F11), Pacific Blue anti-CD90 (Clone 30.H12), APC anti-EpCam (Clone G8.8), Alexa Fluor 594 anti-vimentin (Clone W1622A), APC anti-CD140a (Clone APA5), Alexa Fluor 647 anti-CD74 (Clone In1/CD74), PE anti-CD49a (Clone HMα1), PE Cy7 anti-F4/80 (Clone BM8) and PerCP Cy5.5 anti-CD3 (Clone 17A2) (Biolegend, San Diego, CA). Cells were then analyzed with an LSR Fortessa cytometer (BD Biosciences, San Jose, CA). Citrine⁺ cells were sorted with a Sony SH800S supported by NIH S10OD023410.