Black walled 96 well plates

CD15+ neutrophils were seeded at 20,000 cells per well in in HBSS with or without 15 μM Cytochalasin D (Sigma Aldrich) black walled 96 well plates (Thermo Fisher Scientific) for 1 hour to allow for attachment. Cells were then infected with SARS-CoV-2 at 2 MOI (80 μl at 5x10^5 PFU/ml) for 4 hours. Cells were then washed 2 x with PBS and fixed in 4% PFA. Cells were stained for SARS-CoV-2 RNA via RNAScope and DAPI as per the manufacturer’s instructions. Whole wells were supplemented with 50 μl of PBS post staining and well were scanned on the DMi8 fluorescent microscope (Leica, Buffalo Grove, IL). Total cell number was determined by total frequency of DAPI particles and infected cells determined by SARS-CoV-2 particle signal in proximity to DAPI. Images were analyzed with ImageJ software 1.52n (National Institute of Health, Bethesda, MD).

In total, 591 human ERF-coding genes were selected from ChromoHub.³⁰ (link) All siRNAs targeting these genes were obtained from Dharmacon (Thermo). The siRNA screening of cell migration-related genes was performed as previously described.⁵⁵ (link) Briefly, MCF-7 cells were plated at 12 × 10³ cells per well in black-walled 96-well plates (Nunc) in antibiotic-free growth medium (HyClone) 12 h before the transfection. The transfections were performed robotically using a Handler workstation SX15 with siRNAs (final concentration 25 nM) and Lipofectamine RNAiMAX (Thermo) transfection reagent (0.25 μL per well) diluted in Opti-MEM (Thermo).
When cell confluence reached 90% after the transfection, the cells were wounded by generating a longitudinal scratch under 5% variation using a robotically driven (Seiko) stainless-steel pin programmed to deliver a scratch of 0.75 × 4 mm. After wounding, the cells were washed once with growth medium and further incubated for 16 h. During this period, the real-time gap distances were imaged and determined using an IncuCyte high-throughput screening system.

The antioxidant assay was performed using black-walled 96-well plates (Nunc, Roskilde, Denmark) and an Infinite^® M200 micro plate reader (Tecan, Grödig, Austria). Each well with a final volume of 200 μL. 10 mM phosphate buffer (pH 7.4) was used to prepare 1 μM fluorescein and 250 mM AAPH solutions. Each well received 150 μL of fluorescein solution and 25 μL of phosphate buffer, Trolox^® solutions or sample solution to measure the blank, the curve or the samples respectively. The plate was placed into the microplate reader and after 30 min of incubation at 37 °C, 25 μL AAPH solution were added to each well and fluorescence was recorded every 5 min for 120 min using an excitation wavelength of 485 nm and an emission wavelength of 520 nm. ORAC values were calculated using the difference in Areas Under the Fluorescein Decay Curve (AUC) between the blank and a sample. The results were expressed as µmol of Trolox^®/100 g dry weight.