70 mm cell strainer

Spleens removed from the abdominal cavity were placed in ice-cold RPMI-1640 with 10% FBS (RPMI-10). Each spleen was sliced into small pieces and placed onto a 70-mm cell strainer (Corning Life Sciences) attached to a 50-mL conical tube. The excised tissue was then pressed through the strainer using the plunger end of a 5-mL syringe, followed by washes with cold RPMI-10. Cells were resuspended in RPMI-10 with 200 U/mL of DNase 1 (STEMCELL Technologies) and incubated at room temperature for 15 minutes. Cells were then centrifuged at 400 Â g for 5 minutes at 4 C and resuspended in 3 mL of ACK lysing buffer. After 5-minute incubation at room temperature, the reaction was quenched with RPMI-10 and cells were centrifuged at 400 Â g for 5 minutes at 4 C. Cells were then passed through 70-mm cell strainer to remove tissue debris, spun at 400 Â g for 10 minutes, and resuspend in flow staining buffer (PBS with 10% FBS and 0.09% NaN 3 ) using 5 mL per spleen. Cells were then used in downstream applications.

To isolate splenic immune cells, murine spleens were cut into small pieces and digested with 0.1 mg/mL collagenase D (Sigma-Aldrich, 11088866001) and 0.05 mg/mL DNase (Sigma-Aldrich, D5025-150KU) for 10 minutes at 37 C. EDTA (Applichem, A4892.1000) was added at a concentration of 0.01 mol/L, followed by a second incubation step at 37 C for 5 minutes. The splenocytes solution was smashed through a 70-mm cell strainer (Corning, 431751). Red blood cells were lysed with cell lysis buffer (BD, 555899). Mouse CD11c UltraPure microbeads (Miltenyi Biotec, 130-108-338), mouse CD8a þ T Cell Isolation Kit (Miltenyi Biotec, 130-104-075) or the Mouse B Cell Isolation Kit (Miltenyi Biotec, 130-090-862) were used according to the manufacturer's instructions to isolate different immune cell populations from the splenocytes suspension. For subsequent in vitro assays with splenic immune cells, cells were resuspended in R10 (RPMI1640; Gibco, 31870-025) supplied with 10% FBS (Gibco, 16140), 1% penicillin-streptomycin (P/S; Gibco, 11548876), 1% L- glutamine (Gibco, 25030-024), 1% sodium-pyruvate (Gibco, 11360-039), 1% nonessential amino acids (Gibco, 11140-035), and 50 mmol/L b-Mercaptoethanol (Gibco, 31350-010).

Mice were mostly sacrificed by CO2 asphyxiation. When lungs were harvested for analysis, mice were instead sacrificed by an intraperitoneal overdose of sodium pentobarbital. Organs were removed and processed according to the following procedure. Lungs were digested for 45 min at 37 C in IMDM medium (Life Technologies) containing 2 mg/ml of type IV collagenase (Worthington) and 0.02 mg/ml DNaseI (Sigma-Aldrich). All other organs were directly disrupted and passed through a 70 mm cell strainer (Corning). Bone marrow cells were flushed from femurs and tibia, and then directly passed through the 70 mm cell strainer. ACK buffer (homemade) was used for erythrocyte lysis for all organs.