Collagenase 4 dnase 1 mix

Mice were euthanized at 11 days post-infection. Before obtaining the lung tissue, mice were perfused with sterile saline to remove the blood. The excised lung tissue was cut into small pieces and incubated in 5 ml of digestion buffer (collagenase IV/DNase I mix, Invitrogen Corporation) for 30 min at 37 °C. Digested lung tissue was pressed through a 200-gauge stainless-steel mesh and was then suspended in Hank’s balanced salt solution (HBSS). Lymphocytes were isolated using mouse lymphocyte separation medium (Dakewe Biotech) and density gradient centrifugation. The isolated cells were washed twice in HBSS and resuspended in complete RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin. After the lymphocytes were isolated, the cells were calculated by the blood cell counting plate with trypan blue staining.

Mice were sacrificed at different time points after malaria infection. The liver, lung, blood, spleen, and peripheral blood mononuclear cell (PBMC) were collected, firstly. Then lung was cut to small pieces and incubated in 5 ml of digestion buffer (collagenase IV/DNase I mix, Invitrogen Corporation) for 30 min at 37 °C. The digested lung tissue was pressed through 200-gauge stainless-steel mesh, and then was suspended in Hank’s balanced salt solution (HBSS). Liver, lung, spleen, and mesenteric lymph nodes (MLN) were mechanically dissociated and processed through a 100-μm cell strainer (BD Falcon), and suspended in HBSS. Lymphocytes were isolated by Ficoll-Hypaque (DAKEWE) density gradient centrifugation. Isolated cells were washed twice in HBSS and re-suspended at 2×10⁶ cells/ml in complete RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine, and 50 μM 2-mercaptoethanol.