Mneongreen

Manufactured by Agilent Technologies

MNeonGreen is a fluorescent protein derived from the sea anemoneEmonin. It exhibits bright green fluorescence with an excitation maximum at 506 nm and an emission maximum at 517 nm.

Automatically generated - may contain errors

Lab products found in correlation

Calpain inhibitor 3, by Cayman Chemical (2 mentions) Pfi 3, by Cayman Chemical (2 mentions) Celltiter glo kit, by Promega (1 mentions)

3 protocols using mneongreen

High-throughput SARS-CoV-2 Antiviral Screening

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Calpain Inhibitor III (#14283), SIS3 (#15495), APY0201 (#9001589), and PFI-3 (#15267) were purchased from Cayman Chemical. Drugs were resuspended at a stock concentration of 40mM in DMSO and then two-fold serial dilutions were performed in DMSO. 20 nanoliters of 1000X drug stock were spotted into each well of a 384-well plate using Labcyte ECHO acoustic dispenser at the Yale Center for Molecular Discovery. 1.25 × 10³ Vero-E6 cells were plated per well in 20 μl of phenol-red free DMEM containing 5% FBS. Two days later, 5,000 PFU (MOI ~1) icSARS-CoV-2-mNG in 5 μl media was added. Cells were incubated at 37°C and 5% CO2 for two days. Infected cell frequencies were quantified by mNeonGreen at 2 dpi (Cytation 5, BioTek). In parallel, 500 PFU (MOI~0.2) SARS-CoV-2 in 5 μl media was added to replicate plates and cell viability was quantified by CellTiter Glo at 3 dpi.

Wei J., Alfajaro M.M., Hanna R.E., DeWeirdt P.C., Strine M.S., Lu-Culligan W.J., Zhang S.M., Graziano V.R., Schmitz C.O., Chen J.S., Mankowski M.C., Filler R.B., Gasque V., de Miguel F., Chen H., Oguntuyo K., Abriola L., Surovtseva Y.V., Orchard R.C., Lee B., Lindenbach B., Politi K., van Dijk D., Simon M.D., Yan Q., Doench J.G, & Wilen C.B. (2020). Genome-wide CRISPR screen reveals host genes that regulate SARS-CoV-2 infection. bioRxiv.

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Evaluating SARS-CoV-2 Mutant Sensitivity to Remdesivir

Cited 1 time

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Each icSARS-CoV-2 mutant virus (E802D and E802A) was cultured in the presence of increasing concentrations of RDV, ranging from 0.39 to 40 μM. Infections were performed using Vero-E6 in 384-well plates containing the corresponding RDV dilutions in phenol-red free DMEM media with 5% FBS and incubated at 37 °C for 4 h. Next, cells were infected with 0.01 MOI of each mutant virus and infected cell frequencies were measured at 48 h post-infection by mNeonGreen expression by high content imaging (Cytation 5, BioTek)^{27 (link)}. Cell culture supernatants were also collected for plaque assay. Cell viability in uninfected cells was assessed at 72 h post-infection using the CellTiter-Glo kit (Promega) according to the manufacturer’s instructions.

Gandhi S., Klein J., Robertson A.J., Peña-Hernández M.A., Lin M.J., Roychoudhury P., Lu P., Fournier J., Ferguson D., Mohamed Bakhash S.A., Catherine Muenker M., Srivathsan A., Wunder EA J.r., Kerantzas N., Wang W., Lindenbach B., Pyle A., Wilen C.B., Ogbuagu O., Greninger A.L., Iwasaki A., Schulz W.L, & Ko A.I. (2022). De novo emergence of a remdesivir resistance mutation during treatment of persistent SARS-CoV-2 infection in an immunocompromised patient: a case report. Nature Communications, 13, 1547.

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SARS-CoV-2 Infection Inhibition Assay

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Calpain Inhibitor III (#14283), SIS3 (#15495), and PFI-3 (#15267) were purchased from Cayman Chemical. Drugs were resuspended at a stock concentration of 40 mM in DMSO and then two-fold serial dilutions were performed in DMSO. 20 nL of 1000X drug stock were spotted into each well of a 384-well plate using Labcyte ECHO acoustic dispenser at the Yale Center for Molecular Discovery. 1.25 × 10³ Vero-E6 cells were plated per well in 20 μl of phenol-red free DMEM containing 5% FBS. Two days later, 5,000 PFU (MOI ∼1) icSARS-CoV-2-mNG in 5 μl media was added. Cells were incubated at 37°C and 5% CO2 for two days. Infected cell frequencies were quantified by mNeonGreen at 2 dpi (Cytation 5, BioTek). In parallel, 1000 PFU (MOI∼0.2) SARS-CoV-2 in 5 μl media was added to replicate plates and cell viability was quantified by CellTiter Glo at 3 dpi. Vero-E6, Huh7.5 and Calu-3 cells were pretreated with 10 μM SIS3 and 40 μM PFI-3 for 48 hours and then infected with SARS-CoV-2 at a MOI of 0.1. Viral production was determined by plaque assay. Cytotoxicity was not observed in these cell lines during the time and concentration of drug used.

Wei J., Alfajaro M.M., DeWeirdt P.C., Hanna R.E., Lu-Culligan W.J., Cai W.L., Strine M.S., Zhang S.M., Graziano V.R., Schmitz C.O., Chen J.S., Mankowski M.C., Filler R.B., Ravindra N.G., Gasque V., de Miguel F.J., Patil A., Chen H., Oguntuyo K.Y., Abriola L., Surovtseva Y.V., Orchard R.C., Lee B., Lindenbach B.D., Politi K., van Dijk D., Kadoch C., Simon M.D., Yan Q., Doench J.G, & Wilen C.B. (2020). Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection. Cell, 184(1), 76-91.e13.

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