Eclipse ts2 microscope

Migration assays were made with Transwell migration chambers (Costar, 8 μm pore size; Sigma-Aldrich, St. Louis, MO) and the vendor’s procedures. For each lung cancer cell line 8 × 10⁴ cells were plated onto individual wells. After 18 hours incubation for human and 24 hours for murine lung cancer cell lines fixation was with 70% methanol, stained with crystal violet and counted using a Nikon Eclipse ts2 microscope.
Invasion assays were with matrigel invasion chambers (Corning, Corning, NY) following the manufacturer’s methods. After 18 hours incubation for human and 24 hours for murine lung cancer cell lines fixation was with 70% methanol, staining was with crystal violet, and counting was with a Nikon Eclipse ts2 microscope. The assays were in triplicate with at least three independent biological replicates.

One day before infection, VeroE6/TMPRSS2 cells and Calu-3 cells were seeded into a 12-well plate. SARS-CoV-2 was inoculated at m.o.i. = 0.1 and incubated at 37 °C for 1 h. The infected cells were washed, and 1 ml of culture medium was added. The culture supernatant (100 µl) and cells were collected at the indicated time points. The samples were subjected to analyze viral titer shown as TCID₅₀ and viral RNA copy number quantified by RT-qPCR (see below). To monitor the syncytial formation in infected cell culture, bright-field photos were obtained using an Eclipse Ts2 microscope (Nikon).
The infection experiment using human iPSC-derived alveolar epithelial cells was performed as previously described^{10 (link)}. Briefly, the working viruses were diluted with Opti-MEM (Thermo Fisher Scientific, Cat# 11058021). Infection experiments were done with cells at an air-liquid interface. The diluted viruses (1000 TCID₅₀ in 100 μl) were inoculated onto the apical side of the culture and incubated at 37 °C for 1 h. The inoculated viruses were removed and washed twice with Opti-MEM. To harvest the viruses on the apical side of the culture, 100 μl of Opti-MEM was applied onto the apical side of the culture and incubated at 37 °C for 10 min. The Opti-MEM applied was harvested and used for RT-qPCR to quantify the viral RNA copy number (see below).