Supernatants from SARS-CoV-2 infected brain organoids were collected at 16 h post-infection and stored at -80°C until used. 600,000 Vero E6 cells were seeded overnight at 37°C / 5% CO2 in 12-well plates. Confluent Vero E6 cells were then washed once with 1xPBS and infected with 10-fold serial dilutions of the collected supernatants. Cells were incubated with the virus for 1 h at room temperature, followed by inoculum removal and addition of 1ml overlay media (2xMEM and 2.5% Avicel (FMC BioPolymer, RC-591 NF) at a 1:1 ratio). 2xMEM contains 100 ml 10x MEM (Gibco), 10 ml 100x penicillin-streptomycin (Fisher Scientific), 10 ml 100x L-Glutamine, 6 ml 35% BSA, 10 ml 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Gibco), 24 mL 5% NaHCO3 (Gibco) and 340 ml water. Plates were incubated 3 days at 37°C, 5%CO2, and then fixed and stained using 0.1% Crystal Violet and 5% PFA (Boston BioProducts) overnight at 4°C. Plaques were quantified and recorded as PFU/ml.
Crystal violet
Manufactured by Boston BioProducts
Crystal Violet is a synthetic dye commonly used in microbiology as a stain. It has the chemical formula C₂₅H₃₀ClN₃. The dye binds to bacterial cell walls and nucleic acids, allowing visualization under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Avicel, by FMC Biopolymer (2 mentions)
Nahco3, by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde (pfa), by Boston BioProducts (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (2 mentions)
4 2 hydroxyethyl 1 piperazineethanesulfonic acid hepes, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
2 protocols using crystal violet
1
SARS-CoV-2 Plaque Assay in Vero E6 Cells
2
SARS-CoV-2 Viral Growth Quantification
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To evaluate SARS-CoV-2 viral growth, the amount of released infectious particles was measured by plaque assay. Briefly, supernatants from SARS-CoV-2 infected cells were collected at indicated time points and stored at −80°C until used. 600,000 Vero E6 cells were seeded and incubated overnight at 37°C / 5% CO2 in 12-well plates. Confluent Vero E6 cells were then washed once with 1xPBS and infected with 100 μl of virus-containing supernatants that were serially diluted 1:10. Plates were incubated 1 h at room temperature, followed by inoculum removal and addition of 1ml overlay media (2xMEM and 2.5% Avicel (FMC BioPolymer, RC-591 NF) at 1:1 ratio). 2xMEM contains 100 mL 10x MEM (GIBCO), 10 mL 100x penicillin-streptomycin (Fisher Scientific), 10 mL 100x L-Glutamine, 6 mL 35% BSA, 10 mL 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, GIBCO), 24 mL 5% NaHCO3 (GIBCO) and 340 mL water. Plates were incubated 3 days at 37°C, 5% CO2, and then fixed and stained using 0.1% Crystal Violet and 5% PFA (Boston BioProducts) overnight at 4°C.
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