Isolation of CD45+ Cells from Tissue

Samples were washed with calcium-free and magnesium-free phosphate-buffered saline and then separated into thin layers and cut into small pieces on ice. The tissues were then digested using an enzyme cocktail, which consisted of 3 mg/ml collagenase type I (Sigma–Aldrich), 0.156 mg/ml collagenase type XI (Sigma–Aldrich), 0.25 mg/ml soybean trypsin inhibitor (Worthington), 0.1875 mg/ml lyophilized elastase (Worthington), 0.24 mg/ml hyaluronidase type I (Sigma–Aldrich) and 60 U/ml DNase I (Sigma–Aldrich) (6 (link), 13 (link)). Tissues were digested in a 37°C water bath for 30 min, the supernatants were collected, with Dulbecco's modified Eagle's medium (DMEM) and 10% fetal bovine serum added to stop the reaction. Fresh enzyme cocktail was then added to the digested tissues for a second round of digestion. The digestion process was repeated 3–4 times. Then, the erythrocytes were removed via ACK lysis buffer (Lonza) treatment for 5 min. The CD45⁺ cells were then purified using a magnetic cell sorting system (Miltenyi Biotec) according to the manufacturer's instructions. Following centrifugation (300 g, 4°C, and 5 min), the cell pellet was collected. Finally, 10 μl of suspension was counted under an inverted microscope using a hemocytometer. Trypan blue was used to quantify live cells.