HIV-1 infected cells maintained in complete medium were washed twice with HBSS containing Calcium/Magnesium (Thermo Fisher Scientific) and resuspended at 1 × 106 cells/mL in the fresh complete medium. The cells were chilled on ice for 1 h before use. One hundred microliters of the medium containing 230 nM of CPP-HuscFvs, 10 nM of protease inhibitor, Darunavir, or 10 μM of nucleoside reverse transcriptase inhibitor, Tenofovir, were placed into appropriate wells of a 96-well Cell Culture Plate (Eppendorf AG). To each well was then added 100 μL of the infected cells and the plate was incubated at 37 °C in a CO2 incubator for 24 h. The cells were removed from the virus-containing culture medium by centrifugation at 250× g, 4 °C for 10 min. The supernatant (180 μL) was transferred to new microcentrifuge tube and then re-centrifuged at 8000× g, 4 °C for 10 min to remove cell debris. The clear supernatant was used subsequently in three different experiments including: HIV-1 viral load assay (5 μL of the supernatant were diluted 200-fold with DPBS without Calcium/Magnesium (Thermo Fisher Scientific); HIV-1 RT enzymatic activity assay (60 μL of the supernatant); and HIV-1 virus infection assay (50 μL of the supernatant). All portions of the supernatant were kept at −80 °C until use.
Calcium magnesium
Manufactured by Thermo Fisher Scientific
The Calcium/Magnesium lab equipment is designed to measure the concentrations of calcium and magnesium in various samples. It utilizes analytical techniques to quantify the levels of these two essential minerals.
Automatically generated - may contain errors
Lab products found in correlation
Coelenterazine h, by Promega (2 mentions)
Freestyle 293 suspension media, by Thermo Fisher Scientific (2 mentions)
Dynasore, by Merck Group (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Cell culture plate, by Eppendorf (1 mentions)
Phenformin, by Merck Group (1 mentions)
Selumetinib, by Selleck Chemicals (1 mentions)
Eeyarestatin 1, by Cayman Chemical (1 mentions)
Nms 873, by Selleck Chemicals (1 mentions)
Vemurafenib, by Selleck Chemicals (1 mentions)
Chloroquine diphosphate, by Merck Group (1 mentions)
6 protocols using calcium magnesium
1
Quantifying HIV-1 Viral Load and Infection
2
BRET Assay with Suspension Cells
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BRET assays were performed and analyzed as previously described 4 (link) with the following modifications: HEK-293S cells grown in FreeStyle 293 suspension media (Thermo Fisher) were transfected at a density of 1 million cells/mL in 2 mL volume using 600 ng total DNA at 1:1 ratio of Receptor-rLuc:Nb6-mVenus and a DNA:PEI ratio of 1:5, and incubated in a 24 deep well plate at 220 rpm, 37°C for 48 hours. Cells were harvested by centrifugation, washed with Hank's Balanced Salt Solution (HBSS) without Calcium/Magnesium (Gibco), and resuspended in assay buffer (HBSS with 20 mM HEPES pH 7.45) with 1 μg/mL freshly prepared coelenterazine h (Promega). Cells were plated in white-walled, white-bottom 96 well plates (Costar) in a volume of 60 μl/well and 60,000 cells/well. Ligands were prepared in drug buffer (assay buffer with 0.1% BSA, 6 mM CaCl2, 6 mM MgCl2), and added at a 1:2 ratio of drug:cell suspension. Ten minutes after the addition of ligand, plates were read using a SpectraMax iD5 plate reader using 485 nm and 535 nm emission filters with a one-second integration time per well. The computed BRET ratios (mVenus/RLuc emission) were normalized to ligand-free control (Net BRET) prior to further analysis.
3
BRET Assay with Suspension Cells
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BRET assays were performed and analyzed as previously described 4 (link) with the following modifications: HEK-293S cells grown in FreeStyle 293 suspension media (Thermo Fisher) were transfected at a density of 1 million cells/mL in 2 mL volume using 600 ng total DNA at 1:1 ratio of Receptor-rLuc:Nb6-mVenus and a DNA:PEI ratio of 1:5, and incubated in a 24 deep well plate at 220 rpm, 37°C for 48 hours. Cells were harvested by centrifugation, washed with Hank's Balanced Salt Solution (HBSS) without Calcium/Magnesium (Gibco), and resuspended in assay buffer (HBSS with 20 mM HEPES pH 7.45) with 1 μg/mL freshly prepared coelenterazine h (Promega). Cells were plated in white-walled, white-bottom 96 well plates (Costar) in a volume of 60 μl/well and 60,000 cells/well. Ligands were prepared in drug buffer (assay buffer with 0.1% BSA, 6 mM CaCl2, 6 mM MgCl2), and added at a 1:2 ratio of drug:cell suspension. Ten minutes after the addition of ligand, plates were read using a SpectraMax iD5 plate reader using 485 nm and 535 nm emission filters with a one-second integration time per well. The computed BRET ratios (mVenus/RLuc emission) were normalized to ligand-free control (Net BRET) prior to further analysis.
4
Radioiodide Uptake Assay Drugs
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Drugs used to treat cells in radioiodide uptake assays are listed in the key resources table . Chloroquine diphosphate was resuspended in PBS without calcium/magnesium (Thermo Fisher); Phenformin HCL in 100% ethanol and all other drugs in dimethyl sulfoxide (DMSO; Sigma-Aldrich), before being diluted in RPMI-1640 medium (Life Technologies) and added to cells at 1:100 dilution. Drugs reported to enhance NIS function and used as positive controls were DBeQ (Sigma-Aldrich), Dynasore (Sigma-Aldrich), Eeyarestatin-1 (Cayman Chemicals), NMS-873 (SelleckChem), Selumetinib (SelleckChem) and Vemurafenib (SelleckChem).
5
Preparation and Administration of Antiviral Drugs
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Chloroquine diphosphate (Sigma-Aldrich) was resuspended in PBS without calcium/magnesium (ThermoFisher). vorinostat (SAHA; Stratech Scientific) and Dynasore (Sigma-Aldrich) were resuspended in dimethyl sulfoxide (DMSO; Sigma-Aldrich). All drugs were diluted in RPMI-1640 medium (1:100; Life Technologies) prior to treatment of cells. For intraperitoneal administration (IP) in mice, SAHA was formulated in 5% DMSO, 40% PEG400, 5% Tween-80 and PBS.
6
HIV-1 Infection and Cell Culture
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HIV-1DA5 (containing 1 × 109 copies) was added to the 1 × 106 H9 cells maintained in complete medium and incubated in CO2 incubator at 37 °C for 24 h. The cells were then washed with Hank’s Balanced Salt Solution (HBSS) containing Calcium/Magnesium (Thermo Fisher Scientific) by centrifugation, replenished with fresh complete medium and incubated further. Virus particles in spent culture medium were determined at 24 h-intervals for 15 days by using HIV Ag/Ab Test Kit (Fujirebio, Tokyo, Japan). The infected cells collected at day 15 of infection were cryopreserved in small portions. The percentage of virus-producing cells was checked by measuring intracellular HIV-1 p24 from 1 × 106 infected cell aliquots.
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