Ritonavir

Manufactured by Merck Group

Sourced in United States, Germany

Ritonavir is a pharmaceutical product developed by Merck Group. It is a protease inhibitor used in the treatment of HIV infection. The core function of Ritonavir is to prevent the human immunodeficiency virus (HIV) from multiplying in the body by inhibiting the activity of the HIV protease enzyme.

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Lopinavir/ritonavir (1 citations)

26 protocols using ritonavir

Ritonavir Modulates PBMC Responses

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PBMCs of healthy individuals and UC patients were isolated. The cell pellet was resuspended in RPMI (Thermo Fisher Scientific, Waltham, MA, USA) at a concentration of 1×10⁶ cells/ml. Additionally, 500 µl RPMI with 10% FCS and 1% penicillin-streptomycin (Sigma-Aldrich, St Louis, MO, USA) were added to each well and sample. Wells containing PBMCs and RPMI or PBMCs, RPMI and anti-CD3 (1 µg/ml, BioLegend, San Diego, CA, USA) served as negative and positive controls, respectively. ritonavir was dissolved in 100% ethanol (10 mg/ml). For analysis of the effect of ritonavir, 100 µg/ml ritonavir (Sigma-Aldrich, Deisenhofen, Germany) was added. Cells were incubated for 72 h. The content of each well was centrifuged at 1400 g for 5 min. The pellet was resuspended in FACS buffer and labeled for flow cytometry.
To differentiate between live and dead cells, staining with Zombie Green™ Fixable Viability Kit (BioLegend) was performed according to the manufacturer's instructions.

Jodeleit H., Al-Amodi O., Caesar J., Villarroel Aguilera C., Holdt L., Gropp R., Beigel F, & Siebeck M. (2018). Targeting ulcerative colitis by suppressing glucose uptake with ritonavir. Disease Models & Mechanisms, 11(11), dmm036210.

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Evaluating Anti-HIV and Anti-Alzheimer's Compounds

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Zidovudine, abacavir, lamivudine, efavirenz, nevirapine, ritonavir, and nelfinavir were obtained from Sigma. Emtricitabine, tenofovir disoproxil fumarate, darunavir, dolutegravir and elvitegravir were obtained from MedChem Express. LysoSensor (DN160) was obtained from Fisher Thermo Scientific. Aβ_1–40 and Aβ_1–42 ELISA kits were obtained from Wako. ML-SA1 was obtained from Tocris Bioscience.

Hui L., Ye Y., Soliman M.L., Lakpa K.L., Miller N.M., Afghah Z., Geiger J.D, & Chen X. (2019). Antiretroviral drugs promote amyloidogenesis by de-acidifying endolysosomes. Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology, 16(1), 159-168.

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Measuring T-cell Glycolytic Response to Activation

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Baseline ECAR of CD4⁺ and CD8⁺ T-cells was determined for roughly 1 h prior to injection of GLUT1/4 inhibitor ritonavir (20 µM; Sigma). A 40-min period of incubation with the inhibitor occurred prior to injection of αCD3/28 as above. Corresponding ECAR was monitored for 4 h after αCD3/28 injection. A final injection of 2-DG (100 mM) arrested glycolysis. Fold ECAR change was calculated by dividing the 13 measurements post antibody injection (dashed box) by the 13 measurements pre antibody injection (dotted box).

Jones N., Cronin J.G., Dolton G., Panetti S., Schauenburg A.J., Galloway S.A., Sewell A.K., Cole D.K., Thornton C.A, & Francis N.J. (2017). Metabolic Adaptation of Human CD4+ and CD8+ T-Cells to T-Cell Receptor-Mediated Stimulation. Frontiers in Immunology, 8, 1516.

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Metabolic Profiling of Cell Lines

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Oxygen consumption and extracellular acidification rates, OCR and ECAR respectively, were measured on an XF24 apparatus from Seahorse Biosciences, as described previously (25). Briefly, all experiments were carried out in filter-sterilized DMEM reconstituted from powder formulation (Sigma, St. Louis, MO, USA) supplemented with 1 mM glutamine, but without sodium bicarbonate, phenol red, or serum, at a pH between 7.2 and 7.4 at 37 °C. Cells were seeded at a density of 1.5 to 2.0 × 10⁵ per well by centrifugation in XF24 culture plates that were coated with poly-d-Lysine (Sigma). Glucose, 2-deoxyglucose (Sigma) and glucose transporter inhibitors (STF-31, GTI-2, and WZB117 from Merck; ritonavir and indinavir from Sigma) were injected to the indicated final concentrations. In each experiment, 6 wells were dedicated to each inhibitor concentration, 4 for dimethyl sulfoxide (DMSO) controls and 2 for background detection; titrations over six different concentrations were therefore conducted on two separate XF24 plates and cartridges for each donor. Data are represented as the average with error bars indicating the standard deviation.

Kavanagh Williamson M., Coombes N., Juszczak F., Athanasopoulos M., Khan M.B., Eykyn T.R., Srenathan U., Taams L.S., Dias Zeidler J., Da Poian A.T, & Huthoff H. (2018). Upregulation of Glucose Uptake and Hexokinase Activity of Primary Human CD4+ T Cells in Response to Infection with HIV-1. Viruses, 10(3), 114.

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Antiretroviral Drugs and Novel PI Evaluation

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The antiretroviral drugs used in this study included zidovudine (ZDV, AZT), lamivudine (3TC), indinavir (IDV), nelfinavir (NFV), saquinavir (SQV), and ritonavir (RTV), all from Sigma-Aldrich Co. (St. Louis, MO, USA); and didanosine (ddI), stavudine (d4T), nevirapine (NVP), and efavirenz (EFV), all from Shanghai Desano Chemical Pharmaceutical Development Co., Ltd. (Shanghai, China). All of these drugs are used commonly in China for antiretroviral treatment. DG35 was a new PI, provided by Hesi Scientific and Technology Ltd (Xi’an, Shaanxi, China).
All of these drugs were dissolved in dimethyl sulfoxide (DMSO) and stored at -20°C. Drugs were serially diluted, such that the final concentration of DMSO in cell culture medium was 0.5%.

Chang S., Zhuang D., Guo W., Li L., Zhang W., Liu S., Li H., Liu Y., Bao Z., Han J., Song H, & Li J. (2016). The Antiviral Activity of Approved and Novel Drugs against HIV-1 Mutations Evaluated under the Consideration of Dose-Response Curve Slope. PLoS ONE, 11(3), e0149467.

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Anti-Viral Drug Solubilization Protocol

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Lopinavir, ritonavir, amprenavir, and darunavir were purchased Sigma or Cambridge Biosciences in powder form. The drugs were solubilised in DMSO at a stock concertation of 5 mM concentration and then further diluted to 100 μM in 200 mM ammonium acetate supplemented with 0.5% C₈E₄.

Mehmood S., Marcoux J., Gault J., Quigley A., Michaelis S., Young S.G., Carpenter E.P, & Robinson C.V. (2016). Mass spectrometry captures off-target drug binding and provides mechanistic insights into the human metalloprotease ZMPSTE24. Nature chemistry, 8(12), 1152-1158.

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In Vitro Evaluation of Antiviral Compounds

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For virus culture, HCoV-229E was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) while MRC-5 human lung fibroblast host cells were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea). The QuantiChrom Nitric Oxide Assay Kit and QuantiChrom Peroxide Assay Kit (BioAssay Systems, Hayward, CA, USA) were used to measuring total NO and H₂O₂ levels, respectively, and the alamarBlue Assay Kit (Invitrogen, Waltham, MA, USA) was used to measure cell cytotoxicity. All procedures were performed according to the manufacturers' instructions. Chloroquine diphosphate and ritonavir were purchased from Sigma-Aldrich (Yongin, Korea). Stocks of these drugs were prepared in dimethyl sulfoxide (DMSO) and stored at −20 °C until further use. An antibody specific to the HCoV S protein was obtained from Sino Biological (Chesterbrook, PA, USA). All cell culture plasticware was purchased from SPL Life Sciences (Pocheon, Korea). Nitrogen (99.99%) and oxygen (99.99%) gases were purchased from Dong-A Scientific (Seoul, Korea). Other analytical reagent-grade solvents and chemicals were purchased from Sigma-Aldrich (Merck, St. Louis, MO, USA). Distilled water with a resistivity of 18.2 MΩ.Cm, obtained from a Milli-Q® water purification system (Millipore, Bedford, MA, USA) was used to prepare all aqueous solutions in this study.

Kaushik N.K., Bhartiya P., Kaushik N., Shin Y., Nguyen L.N., Park J.S., Kim D, & Choi E.H. (2022). Nitric-oxide enriched plasma-activated water inactivates 229E coronavirus and alters antiviral response genes in human lung host cells. Bioactive Materials, 19, 569-580.

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Antivirals and Chloroquine Evaluation

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DMSO, dexamethasone (D4902), ribavirin (R9644), lopinavir (SML0491), ritonavir (SML1222), hydroxychloroquine sulphate (HO915), chloroquine diphosphate (C6628), and azithromycin dihydrate (A9834) were supplied by Sigma-Aldrich (Overijse, Belgium). In the case of the latter 3 drugs, the salt forms were purchased to avoid limitations due to the low solubility of the drugs. Remdesivir (30354-10) was purchased from Sanbio (Uden, The Netherlands), and favipiravir (FF29069) was obtained from Biosynth Carbosynth (Compton, United Kingdom). The peptide mimetic ¹⁰Panx1 was synthesised in-house by means of solid-phase peptide synthesis with a purity of >98%. Lanthanum trichloride was supplied by Merck Chemical n.v./s.a. (Overijse, Belgium). All other reagents were obtained from various suppliers at the highest analytical grade possible. Each drug was dissolved in the respective solvent (Table 1).

Caufriez A., Tabernilla A., Van Campenhout R., Cooreman A., Leroy K., Sanz Serrano J., Kadam P., dos Santos Rodrigues B., Lamouroux A., Ballet S, & Vinken M. (2022). Effects of Drugs Formerly Suggested for COVID-19 Repurposing on Pannexin1 Channels. International Journal of Molecular Sciences, 23(10), 5664.

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HIV-1 Gag Protein Processing

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HEK-293 or HeLa cells, optionally transfected with HIV-1 Gag, c-myc-HIV-1 PR and HA-BCA3, were washed with PBS, pelleted by centrifugation at 300 × g for 5 min and frozen at −70°C. The cell pellet was resuspended in 500 μl solution B (60 mM MES, pH 6.5, 120 mM NaCl, 0.1% NP-40) and homogenized by 50 strokes in a Dounce homogenizer. The cell lysate was centrifuged for 10 min at 1 000 × g. The pellet was resuspended in 500 μl 60 mM MES, pH 6.5, containing 120 mM NaCl, homogenized by 30 strokes in a Dounce homogenizer and centrifuged for 10 min at 1 000 × g. Both supernatants were combined and incubated with recombinant HIV-1 PR (1.25 μM final concentration), optionally in the presence of ritonavir (Sigma) (4 μM final concentration) or calpain inhibitor I (Sigma) (33–100 μM final concentration) at 37°C. The reaction was stopped by addition of 2× protein loading buffer (PLB). Samples were separated on 12 or 15% SDS-PAGE and analyzed by Western blotting.

Rumlová M., Křížová I., Keprová A., Hadravová R., Doležal M., Strohalmová K., Pichová I., Hájek M, & Ruml T. (2014). HIV-1 protease-induced apoptosis. Retrovirology, 11, 37.

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Antimicrobial Library Compound Preparation

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The antimicrobial library, consisting of 112 compounds (68 antibacterials; 39 antivirals; and five other) was purchased from Selleck Chemicals (Burlington, ON, Canada) and contained the PIs, NFV, indinavir, amprenavir, and atazanavir. Additional PIs – lopinavir, ritonavir, darunavir (DRV), and saquinavir were purchased from Sigma-Aldrich (St Louis, MO, USA). Compounds were dissolved in dimethylsulfoxide (DMSO) to a stock concentration of 10 mM. Subsequent dilutions were made using cell culture media, as needed, to gain concentrations of 40 to 10⁻⁶ µM.

Meier-Stephenson V., Riemer J, & Narendran A. (2017). The HIV protease inhibitor, nelfinavir, as a novel therapeutic approach for the treatment of refractory pediatric leukemia. OncoTargets and therapy, 10, 2581-2593.

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