Type 2 collagenase

We harvested inguinal lymph nodes at 2 weeks after ACLT and digested the lymph nodes with a mixture of type II collagenase (Yeasen, Shanghai, China) and DNase I (Yeasen, Shanghai, China) for 20 min at room temperature. The digest was filtered through a 70 μm cell strainer (Miltenyi, Germany) and then washed with 1× PBS. For staining of cytokine interleukin 17a (IL-17a), cells were stimulated with the leucocyte activation cocktail BD Pharmingen (BD Biosciences, Franklin Lakes, New Jersey, U.S.) for 4 hours at 37°C. Then, the cells were incubated with antibodies against surface markers on ice for 30 minutes in the dark. After fixation and permeabilization with a BD CytoFix/CytoPerm Kit (BD Biosciences, Franklin Lakes, New Jersey, U.S.), cells were stained with IL-17a fluorescent antibodies on ice for an additional 30 minutes in the dark. The T cell panel included the following antibodies: Zombie NTR Fixable Viability APC-Cy7 (Biolegend, San Diego, California, U.S), CD25 PE-Cy7 (Biolegend), CD3 FITC (Biolegend) and IL-17a PE (Biolegend). Analyses were performed using FlowJo software.

The heart tissue of newborn SD rats was cut into pieces after washing with precold PBS and 40 minutes of digestion with 2.0 g/L type II collagenase (Yeason, China) and 2.5 g/L trypsin (Yeason, China). Then the digest buffer was removed and the samples were cultured with Dulbecco's modified eagle medium (DMEM; Gibco, USA) containing 10% foetal bovine serum (FBS; Gibco, USA), followed by filtering with a 200-mesh copper mesh. The obtained cardiac fibroblasts were then cultivated in a complete medium (37°C, 5% CO₂). For the CF model, cardiac fibroblasts were treated with 100 nM angiotensin II for 24 h to induce an in vitro myocardial fibrosis model.