Uvc 500 ultraviolet crosslinker

RPE cells were exposed to UVC (λ = 254 nm) or to UVB (λ = 302 nm) using Hoefer UVC 500 Ultraviolet Crosslinker (20 J/cm²) or VM25/30/GX trans-illuminator as UV sources, respectively. 1 µM A-1155463 or 0.1% DMSO were added to the cell medium. After 24 h cell viability was measured using a CTG assay (Promega). The luminescence was read with a PHERAstar FS plate reader (BMG Labtech, Ortenberg, Germany).

HeLa parental and genome-edited cell lines were maintained in Dulbecco’s Modified Eagle Medium, supplemented with 10% fetal bovine serum and 2 mM L-glutamine at 37 °C and 5% CO₂. For UVC pulse treatment, cells were seeded in 96-well plates with 100 μL medium, and, after attachment overnight, confluence was monitored at 4 h-intervals in an IncuCyte ZOOM instrument (Essen BioScience) maintained at 37 °C and 5% CO₂. 48 h after seeding, cells were exposed to a UVC pulse (λ = 254 nm, 100 J m⁻²) in a Hoefer UVC 500 Ultraviolet Crosslinker, and confluence was monitored for a total of 160 h. Time points were averaged from eight technical replicates. Control wells on the same plate were exposed to the UVC pulse without removing the plastic cover. For treatment with phleomycin D1, Zeocin (Thermo Fisher Scientific) was added at seeding to a final concentration of 20 μM. When monitoring cell death, SYTOX Green nucleic acid stain (Thermo Fisher Scientific) was added at seeding to a final concentration of 5 μM, and images were taken simultaneously in phase-contrast and green fluorescence modes on an IncuCyte FLR instrument. SYTOX Green nucleic acid stain only penetrates compromised membranes characteristic of dying cells. Green fluorescence was normalized to cell confluence, and the ratio of normalized fluorescence in the absence or in the presence of phleomycin D1 is reported.

SARS-CoV-2 in 120 ml of medium was inactivated with at least 7650 J/m² UVC in a UVC 500 Ultraviolet Crosslinker (Hoefer) using 6 well plates (3 ml per well). Virus was determined to be inactivated by incubation with Vero E6 cells for 4 days without development of CPE. UV-inactive SARS-CoV-2 was then partially-purified using a 20% sucrose cushion centrifuged at 175,000 x g for 3–4 hr using SW32Ti rotor (Beckman Coulter). The pellet was resuspended in PBS and virus washed and concentrated using Amicon Ultra-15 Centrifugal Filter Units with 100 kDa cutoff (Merck Millipore). For infectious SARS-CoV-2 immunization, virus was also concentrated using Amicon Ultra-15 Centrifugal Filter Units with 100 kDa cutoff (Merck Millipore). Mice were injected s.c. in the base of the tail with 100 μl of UV-inactive or infectious SARS-CoV-2 with 25 μg adjuvant in 50 μl made as described previously [119 (link), 120 (link)]. A boost was given 5–6 weeks after prime, and mice were bled to measure serum neutralizing titers 4–9 weeks after boost.