Ra1 lysis buffer

Infected cells in T75 flasks were rocked to swirl up detached cells and the supernatant was harvested. Detached cells were separated from the supernatant by centrifugation. Remaining adherent cells in T75 flasks were trypsinized and afterwards combined with the detached cells from the previous step. For this cell suspension, we then determined cell count and viability (by trypan blue staining) using a Vi-CELL™ (Beckman Coulter). An aliquot of 1 × 10⁶ cells was centrifuged and cell pellets were lysed with 350 μL of lysis buffer RA1 (Macherey Nagel) containing 1 % (v/v) β-mercaptoethanol. Lysates were stored at −80 °C until RNA purification according to the manufacturer’s instructions (“NucleoSpin RNA” from Macherey Nagel). The remaining cell suspension was fixed with paraformaldehyde at a final concentration of 1 % (w/v) and aliquots of 1 × 10⁶ cells were stored in 70 % ethanol at −20 °C until staining for imaging flow cytometry. For infection experiments at low MOI, 2 × 10⁶ cells were collected and fixed for the analysis by imaging cytometry.

In a U-bottom plate, 1 x 10^6 splenocytes/well were seeded in quintuplicate and cultured for 6 hours at 37°C with 5% CO₂ in cRPMI in the presence of inactivated TIGR4 at a cell:bacteria ratio of 1:10. Unstimulated control splenocytes were cultured in cRPMI alone. Upon stimulation, cell replicates were centrifuged at 500 x g for 10 minutes at 4°C. The supernatant was discarded, the pellet resuspended in 50 µl of lysis buffer RA1 (Macherey-Nagel, Germany), flash-frozen in liquid nitrogen, and stored at -70°C for subsequent RNA extraction.

Mesenteric lymph node cells were seeded at 1 × 10⁶ cells/ml 200 μl/well in RPMI-1640 + 10% (v/v) FCS, 50 μM ß-MeSH on a 96 well-plate, and incubated at 37°C, 5% (v/ v) CO₂ for 24 h in the absence or presence of CD3-antibody (EXBIO Praha, Vestec u Prahy, Czech Republic). After the incubation period, cell-free supernatants were generated by centrifugation at 300 g for 10 min at 4°C. Colon tissues were homogenized either for cytokine measurements in ice cold PBS + 1x protease inhibitor cocktail (Cell Signaling Technology, Frankfurt, Germany) or for RNA extraction in lysis buffer RA1 (Macherey Nagel, Düren, Germany) supplemented with 150 mM ß-MeSH using the FastPrep® 24 homogenizer and 2 mL Matrix D FastPrep® tubes (both from MP Biomedicals, Santa Ana, CA, USA) according to the manufacturer's instructions for intestinal tissue.

To isolate RNA from aortic tissue, RA1 lysis buffer (Macherey-Nagel, Düren, Germany) containing 1% DTT was added to the tissue, which was cut in tiny pieces and subsequently minced. RNA was isolated with the RNeasy^® Plus Micro Kit (Qiagen, Hilden, Germany) according to the RNeasy Fibrous Tissue Mini Kit protocol (Qiagen, Hilden, Germany). Isolated RNA (500 ng) was reverse transcribed into cDNA with the qSCript™ cDNA Synthesis Kit (Quanta Biosciences, Beverly, MA) and analyzed by real-time fluorescence assessment of SYBR Green signal (iQ™ SYBR^® Green Supermix, Bio-Rad, Hercules, CA) in the CFX96™ Real-Time Detection System (Bio-Rad, Hercules, CA). Each sample was measured in duplicates. Primers were designed for the murine genes of interest, sequences are listed in Table 1. mRNA levels were analyzed and corrected for the housekeeping gene Hprt. RNA isolation was unsuccessful in one mouse from the 3μg/kg/day OSM group resulting in 6, 7 and 8 mice per group.

hACE2-KI mice (B6.Cg-Ace2^{tm1(ACE2)Dwnt}) and hACE2-K18Tg mice (Tg(K18-hACE2)2Prlmn) were described previously^{3 (link),18 (link)}. All mice were produced at the specific-pathogen-free facility of the Institute of Virology and Immunology (Mittelhäusern), where they were maintained in individually ventilated cages (blue line, Tecniplast), with 12-h:12-h light:dark cycle, 22 ± 1 °C ambient temperature and 50 ± 5% humidity, autoclaved food and acidified water. At least 7 days before infection, mice were placed in individually HEPA-filtered cages (IsoCage N, Tecniplast). Mice (10 to 12 weeks old) were anaesthetized with isoflurane and infected intranasally with 20 μl per nostril with the virus inoculum described in the results section. One day after inoculation, infected hACE2-K18Tg mice were placed in the cage of another hACE2-K18Tg contact mouse. Mice were monitored daily for bodyweight loss and clinical signs. Oropharyngeal swabs were collected under brief isoflurane anaesthesia using ultrafine sterile flock swabs (Hydraflock, Puritan, 25-3318-H). The tips of the swabs were placed in 0.5 ml of RA1 lysis buffer (Macherey-Nagel, 740961) supplemented with 1% β-mercaptoethanol and vortexed. At 2 or 4 dpi, mice were euthanized, and organs were aseptically dissected. Systematic tissue sampling was performed as detailed previously^{3 (link)}.