Cold red blood cell lysis buffer

Mice were euthanized and their spleen were carefully separated and rinsed several times with cold PBS containing 2% FBS. Spleen were ground and passed through a 70 µm cell strainer and the cells were centrifuged at 300 g for 5 min at 4 °C. 10 ml of cold red blood cell lysis buffer (Solarbio) was added, and the samples were incubated for 5 min at 4 °C. The reaction was stopped by adding 20 ml of cold PBS containing 2% FBS and washed once to remove the residual buffer. Lymphocytes were washed once and resuspended in PBS containing 2% FBS.

Lungs were cut into 0.5-cm pieces, placed in gentleMACS C tubes (Miltenyi) containing collagenase type IV (Gibco) and DNase I (Roche) in PBS containing 2% FBS, and dissociated using a gentleMACS Dissociator (Miltenyi; program m_lung_01). A single-cell suspension was obtained by digesting tissue through a 70 µm cell strainer, and centrifugation at 300 g for 5 min at 4 °C. After centrifugation, 1 mL of cold red blood cell lysis buffer (Solarbio) was added for 2 min to lyse red blood cells. The reaction was stopped by adding 10 mL of cold PBS containing 2% FBS and washed once to remove residual red blood cell lysis buffer. Lymphocytes were obtained from the resulting cell suspensions using density gradient centrifugation (Percoll, SIGMA-ALDRICH). Cells were recovered at the interface of the 80% Percoll layer and the 40% Percoll layer, then washed with PBS + 2% FBS at 500 g for 5 min to remove excess Percoll.