SARS-CoV Pathogenesis Is Regulated by a STAT1 Dependent but a Type I, II and III Interferon Receptor Independent Mechanism

Frieman, Matthew B.; Chen, Jun; Morrison, Thomas E.; Whitmore, Alan; Funkhouser, William; Ward, Jerrold M.; Lamirande, Elaine W.; Roberts, Anjeanette; Heise, Mark; Subbarao, Kanta; Baric, Ralph S.

Mice were inoculated as described above, sacrificed by exsanguination at 2 and 4 days post-infection, and lungs were perfused via cardiac puncture with 16 PBS. Lungs were dissected, minced, and incubated for 2 hrs with vigorous shaking at 37uC in digestion buffer [RPMI, 10% FBS, 15 mM HEPES, 2.5 mg/ml collagenase A (Roche), 1.7 mg/ml DNase I (Sigma)]. Cells were passed through a 40 micron cell strainer, resuspended in RPMI media, layered on 5 ml lympholyte-M (Cedarlane), and centrifuged 30 min at 2500 rpm. Cells were collected, washed in wash buffer (16 HBSS, 15 mM HEPES), and total viable cells were determined by trypan blue exclusion. Isolated cells were incubated with anti-mouse FccRII/III (2.4G2; BD Pharmingen) for 20 min on ice and then stained in FACS staining buffer (16 HBSS, 1% FBS, 2% normal rabbit serum) with the following antibodies from eBioscience: anti-F4/80-FITC, anti-Gr-1-PE, anti-CD11b-APC, anti-CD11c-PE, anti-Ly-6C-FITC, anti-CD3-FITC, anti-CD8-APC, anti-CD4-PerCP, and anti-NK1.1-PE. Cells were fixed overnight in 2% paraformaldehyde and analyzed on a Cyan cytometer (Dako) using Summit software.