Flow Cytometric Isolation of Joint Cells

Harvested joints were digested in collagenase/dispase with DNase and shredded with rat-tooth forceps to isolate cells before staining them for flow cytometry as described (22 (link)). About 5x10⁵ cells were incubated in a 96-well U-bottom plate with Fc block (anti-CD16/CD32; eBioscience, San Diego, CA) then surface stained as indicated with the following antibodies: CD45.2 PE, F4/80 APC, Ly-6G PE-Cy7, and Ly-6C FITC (all from eBioscience). Cells were then washed and fixed in 4% paraformaldehyde for 15 minutes. For each sample 50,000 events were analyzed using a BD LSRFortessa X-20 flow cytometer and data analysis was performed using FlowJo 10.8.1 software.