μ dish35 mm quad

1x10⁴ U2OS-ACE2-GFP-1-10 and 1x10⁴ U2OS-ACE2-GFP-11 cells were plated overnight in each compartment of a μ-Dish35 mm Quad (Ibidi). The next day, cells were infected at a MOI of 0.1. 18 hours later, NK cells were added at 1:1 ratio as well as serum from a pre-pandemic or a COVID-19 individual (dilution 1:100). Conditions without NK and Serum (“No serum No NK”) and with NK cells but without serum (“no serum”) were included as controls. Propidium iodide (PI) (10 μg/ml, Invitrogen) was added to monitor cell death. Transmission and fluorescence images were acquired at a 20X magnification every 4 minutes for 4 hours on a BioStation IM-Q (Nikon). At least 5 fields were recorded in each condition. Images were analyzed using the FIJI software.

Cells expressing fluorescently tagged proteins were seeded in DMEM onto 35-mm glass bottom dish (Iwaki) or μ-dish 35 mm Quad (Ibidi) that was pre-coated with 50 μg/mL fibronectin. Cells were observed 3-8 hours after seeding. In the experiments of pharmacological treatments, observation was carried out 5 min after the drug application. The cells were observed using the inverted confocal microscope (A1Rsi, Nikon) equipped with an oil-immersion objective lens (NA 1.49, 60x, Apo-TIRF, Nikon or NA 1.45, 100x, Plan Apo, Nikon) and a heat chamber (TOKAI-HIT) under the 37°C and 5% CO₂ humidified atmosphere.
Acquired images were analyzed using Fiji or NIS-Elements AR (Nikon) software. The average flow speed of myosin IIA-GFP or LifeAct-mCherry in each SF was calculated based on the slope in the kymograph depicted using Multi Kymograph tool in Fiji. This measurement was carried out for three SFs in each cell.