All experiments were performed with TBEV strain Hypr kindly provided by Daniel Růžek (Czech Academy of Sciences, České Budêjovice, Czech Republic). 2’-CMA and 7-deaza-2’-CMA were obtained from Santa Cruz Biotechnology (Dallas, TX, USA), while 2’-CMC and Ribavirin were from Sigma-Aldrich. The test compounds were solubilized in DMSO (sterile filtered, suitable for hybridoma, Sigma-Aldrich) to yield 10 mM concentrated stock solutions.
2 cmc
Manufactured by Merck Group
Sourced in United States
2'-CMC is a chemical compound utilized in various laboratory applications. It functions as a nucleoside analog, specifically targeting the 2' position of the ribose moiety. This compound is commonly employed in research and analytical settings.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Ribavirin, by Merck Group (1 mentions)
Guanosine triphosphate, by Merck Group (1 mentions)
Cytidine 5 triphosphate, by Merck Group (1 mentions)
D2650, by Merck Group (1 mentions)
Rupintrivir, by Merck Group (1 mentions)
Prism 9, by GraphPad (1 mentions)
4 protocols using 2 cmc
1
Antiviral Evaluation of Nucleoside Analogs
2
Hepatitis E Virus Antibody Assay
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2CMC, RBV, guanosine triphosphate (GTP) and cytidine 5’-triphosphate (CTP) were purchased from Sigma-Aldrich and were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St Louis, MO). The HEV-specific antibody was purchased from EMD Millipore (MAB8002).
3
Antiviral Drug Testing on Intestinal Organoids
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United States Pharmacopeia-grade NTZ (Sigma, 1463960) was serially diluted 2-fold in ≥99.7% pure DMSO (Sigma, D2650). ≥95% pure 2’-C-methylcytidine (2’CMC; Sigma, M4949) was serially diluted in 0.5 log10 increments in Milli-Q H2O. Dilutions of compound in their vehicle were then added to 500 μM GCDCA supplemented CMGF- and INTd medium for a 1–2-hour inoculation and 24-hour incubation periods, respectively. NTZ was added to the media with a final concentration of 1% DMSO, and 2’CMC was added to the media with a final concentration of 2% H2O. 100 TCID50 of each virus was added to the inoculation media and then added onto 3 wells of the optimal HIE line and incubated at 37°C. After the incubation period, the HIE monolayers were washed 3 times with CMGF-. The media was replaced with vehicle- or compound-prepared INTd media and cells were incubated at 37°C for 24 hours. Stocks of NTZ were stored at −20°C and not stored longer than 2 weeks.
4
Antiviral Compound Cytotoxicity and Efficacy
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EV-A71-infected HIOs and RD cells were treated with rupintrivir (Sigma-Aldrich, St. Louis, MO, USA), enviroxime (aablocks LLC, San Diego, CA, USA), and 2′CMC (Sigma-Aldrich, St. Louis, MO, USA).
For determination of compounds’ half maximal cytotoxic concentration (CC50), the non-infected HIOs/cells were treated with compounds (0.5–100 μM) for 72 h. The viability % was calculated as follows: (luminescence reading compound treated)/(luminescence reading DMSO control) × 100. The CC50 was calculated using non-linear regression (curve fit) with GraphPad Prism 9 (GraphPad, La Jolla, CA, USA). For determination of compounds half maximal effective concentration (EC50), the infected HIOs/cells were treated with DMSO (i.e., 0 µM) or compounds (0.15–40 μM) for 72 h. The CPE inhibition % was calculated as follows: (luminescence reading compound treated)/(luminescence reading DMSO control) × 100. The EC50 was calculated using non-linear regression (curve fit) with GraphPad Prism 9 (GraphPad).
For determination of compounds’ half maximal cytotoxic concentration (CC50), the non-infected HIOs/cells were treated with compounds (0.5–100 μM) for 72 h. The viability % was calculated as follows: (luminescence reading compound treated)/(luminescence reading DMSO control) × 100. The CC50 was calculated using non-linear regression (curve fit) with GraphPad Prism 9 (GraphPad, La Jolla, CA, USA). For determination of compounds half maximal effective concentration (EC50), the infected HIOs/cells were treated with DMSO (i.e., 0 µM) or compounds (0.15–40 μM) for 72 h. The CPE inhibition % was calculated as follows: (luminescence reading compound treated)/(luminescence reading DMSO control) × 100. The EC50 was calculated using non-linear regression (curve fit) with GraphPad Prism 9 (GraphPad).
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