Phenotypic tropism testing was performed using two methods: (1) Tropism testing in GHOST cell-lines27 (link) of the viruses generated from individual clones (cPTT) and (2) replicative phenotypic tropism test rPhenotyping (pPTT) whereas described previously25 (link). In cPTT co-receptor tropism was determined by measuring Renilla luciferase activity (relative light units [RLU]) using Bright-Glo™ Luciferase Assay System (Promega, US). We consider a 10-fold shift in mean RLU of infected cells over non-infected. The pNL4-3 and pMJ4 were used as positive control for X4- and R5-tropic strains respectively. In addition to luciferase expression, in a subset of clones’ green fluorescent protein (GFP) expression was also captured using confocal microscopy (Olympus Fluoview v2.0b). The rPhenotyping was performed with 31 patients’ samples infected with HIV-1C and four QC-samples. The ligated mixture was transformed into TOP10 bacteria (Life Technologies) and inoculated directly in the LB broth supplemented with ampicillin to retain the viral diversity. The tropism was inferred by using serial dilutions of the CCR5 antagonist TAK-779 (obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, Bethesda, MD, USA) or the CXCR4 inhibitor AMD3100 (Sigma-Aldrich, St. Louis, MO, USA). rPhenotyping was compared with pGTT while cPTT were compared with cGTT.
Tak 779
Manufactured by Merck Group
Sourced in United States
TAK-779 is a lab equipment product designed for research purposes. It functions as a small molecule inhibitor of the CCR5 chemokine receptor, which is involved in cellular signaling pathways. The core function of TAK-779 is to facilitate the study of CCR5-mediated processes in various research models.
Automatically generated - may contain errors
Lab products found in correlation
Amd3100, by Merck Group (2 mentions)
Gsk lsd1, by Merck Group (2 mentions)
Mda mb 468, by American Type Culture Collection (2 mentions)
Mda mb 231, by American Type Culture Collection (2 mentions)
Fluoview v2.0b, by Olympus (1 mentions)
Top10 bacteria, by Thermo Fisher Scientific (1 mentions)
Bright glo luciferase assay system, by Promega (1 mentions)
M0512, by Merck Group (1 mentions)
Minocycline, by Merck Group (1 mentions)
Plx3397, by Apexbio (1 mentions)
P1754, by Merck Group (1 mentions)
Maraviroc, by Merck Group (1 mentions)
Nk cell isolation kit, by Miltenyi Biotec (1 mentions)
Graph pad version 4, by GraphPad (1 mentions)
Mts solution, by Promega (1 mentions)
96 well plate reader, by Tecan (1 mentions)
Dynasore, by Merck Group (1 mentions)
Liveblazer fret b g loading kit, by Thermo Fisher Scientific (1 mentions)
Nh4cl, by Merck Group (1 mentions)
Diphtheria toxin, by Merck Group (1 mentions)
10 protocols using tak 779
1
HIV-1 Coreceptor Tropism Phenotyping
2
Minocycline, TAK-779, and PLX3397 Treatment in Mice
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Minocycline (Sigma-Aldrich) and vehicle (PBS) were administrated i.p. at 10 mg/kg every the other day into mice at P7, and the treated mice were euthanized at P28. TAK-779 (Sigma-Aldrich) was administrated i.p. at 20 mg/kg every day into P7 mice, and the treated mice were euthanized at P28. PLX3397 (ApexBio) was dissolved in DMSO and then added immediately before using to a solution of 0.5% methyl cellulose (M0512; Sigma-Aldrich) and 1.0% Tween 80 (P1754; Sigma-Aldrich). PLX3397 was gavaged into P7 mice at 75 mg/kg every day with a 24G 1-inch stainless steel gavage needle (Braintree Scientific) as described previously (Butchbach et al., 2007 (link)). PLX3397-treated mice were euthanized at P28.
3
Cytotoxic NK Cell Assay
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A total of 2.0 × 104 B16-F10 cells were plated 1 day before the assay on a 48-well plate. NK cells were purified by the NK Cell Isolation Kit (Miltenyi Biotec) and stimulated with 200 U/ml rmIL-2 (402-ML, R&D Systems) for 24 h. Same numbers (2.0 × 104) of activated NK cells were cocultured with B16-F10 cells for 4 h. In order to inhibit CCL5–CCR5 interaction, 0.5 µg/ml of Maraviroc (PZ0002, Sigma-Aldrich), 0.5 µg/ml of TAK779 (SML0911, Sigma-Aldrich) or 0.5 µg/ml anti-mCCL5 (MAB478-100, R&D Systems) antibody were added together with NK cells.
4
Determining IC50 of PKC412 in Cell Lines
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To determine the IC50 for PKC412‐sensitive and PKC412‐resistant cell lines, 1 × 104 cells/well were cultured in 96‐well plates with increasing concentrations of PKC412 (5 to 1000 nm ). Also, cell viability and proliferation at 5 to 1000 nm PKC412 with or without 50 ng/ml CCL5 (PeproTech) and with or without 500 nm TAK‐779 (Sigma‐Aldrich, St. Louis, MO, USA) were measured. After 48 h, 20 μL MTS solution (Promega, Madison, WI, USA) was added to each well and the cells were incubated for an additional 2 h. Absorbance at 490 nm was measured with a 96‐well plate reader (Tecan, Männedorf, Switzerland) in accordance with the manufacturer’s instructions. The relative viability was calculated by dividing the measured value of each well by the measurement of the untreated control. The relative IC50 was calculated by nonlinear regression analysis with graphpad version 4.0 software (GraphPad Software).
5
Virus Entry Fusion Kinetics Assay
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At 24 hr prior to the assay, TZM-bl cells were plated at 4 × 104 cells/well in black clear-bottomed 96 well plates. On the day of assay, cells were cooled on ice prior to the addition of the appropriate MOI of virus (all infections were performed in 100 μL volumes). Immediately following addition of virus harboring Vpr-BlaM, cells were placed at 4°C for 1 hr. Virus was then removed and cells were washed with PBS and 100 μL of DMEMcomp was added to each well before shifting the plate to the 37°C CO2 incubator to initiate viral entry. To gain kinetic data, virus fusion was blocked at the appropriate time point (0, 15, 30, 45, 60, 75, and 90 mins) by removing the media and replacing with media containing Dynasore, TAK 779, NH4Cl, or T20 (Sigma-Aldrich). The inhibitor concentrations were found by testing different concentrations in titration experiments (Supplemental Information ). Note that for the 0 min time point, drugs were added immediately prior to the 37°C temperature shift. After 90 mins, cells were loaded with CCF2-AM from the LiveBLAzer FRET B/G Loading Kit (Life Technologies) and incubated at room temperature in the dark for 2 hr. Finally, the CCF2 was removed; cells were washed with PBS and fixed with 2% PFA prior to viewing.
6
Cell Line Acquisition and Reagents
7
Diphtheria Toxin-Induced Murine Model
8
Cell Line Acquisition and Reagents
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MDA-MB-231, MDA-MB-468 and EMT6 cell lines were from ATCC. 4T1 cell line was provided by Dr. Adrian Lee (University of Pittsburgh). HCI-2509 was purchased from Xcessbio Biosciences Inc. (San Diego, CA). GSK-LSD1 and TAK-779 were obtained from Sigma (St. Louis, MO).
9
HIV gp120 CCR5 Antagonism Assay
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CCR5 blocking was assessed using 1000 ng/mL, 500 ng/mL, and 5 ng/mL of HIV (AD8) recombinant gp120 (CM235) obtained from the NIH AIDS Reagent Repository (Bethesda, MD, United States). CCR5 antagonizing was carried out using 3 μM TAK-779 (Sigma-Aldrich, St. Louis, MO, United States, cat N° 229005-80-5).
10
Polyanionic Carbosilane Dendrimers Evaluation
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Polyanionic carbosilane dendrimers, third-generation G3-S16 with 16 sulfated end groups (C256H508N48Na16O64S16Si29; MW: 6,978.41 g/mol) and second-generation G2-NF16 with naphthylsulfonated end groups (C184H244N24Na16O56S16Si13; MW: 4,934.02 g/mol), were prepared according to reported methods.34 Several reagents and ARV were used as controls: zidovudine (AZT; GSK, GlaxoSmithKline plc, London, UK), enfuvirtide (T-20; Hoffman-La Roche Ltd., Basel, Switzerland), atazanavir (ATV; Bristol-Myers Squibb, New York, NY, USA), and raltegravir (RAL; Merck Millipore, Billerica, MA, USA); Suramin, a polyanionic compound that could mimic the function of dendrimers; CXCR4 chemokine receptor antagonist AMD3100, CCR5 receptor antagonist TAK-779, and colchicine, a microtubule polymerization inhibitor (all from Sigma-Aldrich Co., St Louis, MO, USA).
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