Non-Langendorff method of rat cardiac fibroblast isolation and flow cytometry

Rat cardiac fibroblast cells were isolated from the right and left ventricles and processed for flow cytometry⁶⁶. Upon cutting the rat thoracic cavity to expose the heart, EDTA buffer was injected into the RV through the aorta to freeze contractions. The heart was clamped and removed. EDTA buffer was injected into the apex of the LV. Next, a perfusion buffer was injected into the apex of the LV. Finally, the collagenase buffer was injected into the LV via 3 injections until digestion was apparent. The clamp was removed, and the heart was split into the RV free wall and LV plus septum, respectively, and weighed to measure RV/LV plus septum weight ratio (Fulton index). The RV and LV were separated, manually dissociated, and incubated on tissue culture plates for 20 min at 37 °C to isolate adherent cells. The supernatant containing non-adherent cells was collected and plated on tissue culture plates to ensure maximal collection of adherent cells. The population enriched for non-adherent cells was then collected by centrifugation at 1500 rpm for 5 min and used for downstream analysis. Collected cells were stained with PE-conjugated anti-rat CD3 antibody and FITC conjugated anti-rat CD4 antibody (Biolegend, San Diego, CA) for 1 h and subjected to flow cytometry using BD FACS AriaIIIu flow cytometer. Data were analyzed using FlowJo software v10.0.6.