Most lipids and fluorescent probes were from Avanti Polar Lipids, and DiO, DiI, DiD, and octadecyl rhodamine B chloride (R18) were from Invitrogen. MβCD, NEM, and fluorescein isothiocyanate–, Alexa 647–, or Alexa 555–labeled CTxB were purchased from Sigma-Aldrich. Formaldehyde and DTT were purchased from Avantor and RPI, respectively. Alexa 488–labeled CD4 antibody and Alexa 647–labeled CCR5 antibody were purchased from Novus Biologicals. HIV entry inhibitors (maraviroc and enfuvirtide), SCDase, and PLA2 were purchased from Sigma-Aldrich. HIV fusion peptide was custom-synthesized by the Yale W.M. Keck Biotechnology Resource Laboratory.
Enfuvirtide
Manufactured by Merck Group
Sourced in Belgium
Enfuvirtide is a laboratory product manufactured by Merck Group. It is a synthetic peptide that functions as a fusion inhibitor, preventing the entry of HIV-1 virus into host cells.
Automatically generated - may contain errors
Lab products found in correlation
Amd3100, by Merck Group (2 mentions)
Fluorescein isothiocyanate (fitc), by Merck Group (1 mentions)
Formaldehyde, by Avantor (1 mentions)
Octadecyl rhodamine b chloride r18, by Thermo Fisher Scientific (1 mentions)
Maraviroc, by Merck Group (1 mentions)
Sdf 1, by Merck Group (1 mentions)
Aldrithiol 2 at 2, by Merck Group (1 mentions)
Bms 626529, by Apexbio (1 mentions)
Emtricitabine, by Abcam (1 mentions)
Lamivudine, by Targetmol (1 mentions)
Efavirenz, by Cayman Chemical (1 mentions)
Saquinavir, by Cayman Chemical (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
Polarstar omega plate reader, by BMG Labtech (1 mentions)
Rpmi 1640, by Lonza (1 mentions)
Celltrace far red, by Thermo Fisher Scientific (1 mentions)
Human cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
10 protocols using enfuvirtide
1
Lipid Membrane Fusion Dynamics
2
Pseudotyped Viral Particle Assay
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Viral particles pseudotyped with a murine leukemia virus (MLV) core and the HIV-1 envelope protein were prepared by co-transfection of 293T cells with 3 g pFB-Luc (reporter plasmid), 2 g pHIT60 (MLV-gag-Pol), 1 g MLV gag-mKO (gift of G. Melikyan), and 3 g HIV-1 JRFL Env plasmids per 10 cm culture dish. Viral supernatants were harvested 48 hours after transfection and centrifuged at 2500 rpm for 10 min at 4C. They were flash-frozen and stored at –70 C for later use. Supported lipid bilayers or GUVs composed of bSM/DOPC/Ch (2:2:1) were labeled with 0.1 mol% DiD. The viruses were incubated for 30 min at 55 C in the presence of 10 g/ml enfuvirtide (Sigma, St. Louis, MO) and brought back to room temperature. After equilibration at room temperature, the viruses were added to supported bilayers or GUVs. The heat/enfuvirtide treatment was omitted in control experiments. mKO-labeled HIV pseudoviruses were illuminated through a 540 nm band-pass filter (D540/25, Chroma) and dichroic mirror (565dclp, Chroma) through the objective and fluorescence was observed through a 605 nm band-pass filter (D605/55, Chroma). Supported lipid bilayers and GUVs stained with DiD were illuminated through a 620 nm (ET620/60, Chroma) and via a dichroic mirror (660dclp, Chroma), and observed through a 665 nm band-pass filter (HQ665/60, Chroma).
3
Investigating HIV Strain Infections
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HIV R5 tropic (ADA and BaL) and X4 tropic (IIIB and MN) strains were obtained from Dr. Blankson (JHU). HIV X4 tropic strain HXB2 was provided by the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: pLAI.2, cat 2532 from Dr. Keith Peden, courtesy of the MRC AIDS Directed Program.
For drug treatment studies, cells were pretreated for 30 minutes with 1μM of either Enfuvirtide (T20), or AMD3100 (Sigma-Aldrich). In specified experiments, cells were treated with SDF-1 (Sigma). For degradation experiments, cells were pretreated with 1μM bafilomycin or 50μM chloroquine. Following pretreatment cells were exposed to HIV basolaterally. In specified experiments, HIV was inactivated with 100mM aldrithiol-2 (AT-2, Sigma-Aldrich) prior to exposure.
For drug treatment studies, cells were pretreated for 30 minutes with 1μM of either Enfuvirtide (T20), or AMD3100 (Sigma-Aldrich). In specified experiments, cells were treated with SDF-1 (Sigma). For degradation experiments, cells were pretreated with 1μM bafilomycin or 50μM chloroquine. Following pretreatment cells were exposed to HIV basolaterally. In specified experiments, HIV was inactivated with 100mM aldrithiol-2 (AT-2, Sigma-Aldrich) prior to exposure.
4
Dose-Response Assay for HIV-1 Inhibitors
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We performed a dose–response curve on MoMo30-plant and Enfuvirtide (Sigma SML0934). We used concentrations of MoMo30-plant from 0.314 to 78.25 nM. Stock solution of MoMo30-plant protein (782.5 nM) was diluted to final concentrations of 0.314 nM, 0.609 nM, 1.22 nM, 2.44 nM, 4.88 nM, 9.77 nM,19.55 nM, 39.06 nM,and 78.25 nM. For Enfurvirtide we diluted at stock solution of 782.5 nM to final concentrations of 2.2 nM, 4.43 nM, 8.86 nM, 17.73 nM, 35.44 nM, 70.91 nM, 142.05 nM, 272.73 nM, and 568.00 nM. To each 1 mL of diluted inhibitor we added 5 µL of HIV-1NL43 (equivalent to 1 ng p24) and 10 µL of DEAE. The mixture was than added to 2 × 104 MAGI cells and then incubated for 48 h at 37 °C. and blue cells were counted. The IC50 of MoMo30-plant was determined by curve fitting using the Hill equation and determined using the Dr. Fit program [34 (link)].
5
Pseudotyped Viral Particle Assay
6
Preparation of Small Molecule Stocks
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Small molecules were purchased from ChemBridge (San Diego, CA). Master stocks of the small molecules (with an approximate molecular weight of ∼330 Daltons) were created by dissolving in 100% DMSO at a concentration of 20 mg/ml (∼60,000 μM). All stocks were stored at −20°C. In addition, BMS-626529 (APExBIO; dissolved to 20 mg/mL [∼42,000 μM] in 100% DMSO) and known fusion inhibitor, T20 (enfuvirtide, dissolved in PBS to 5 mg/mL [∼1,110 μM]; Sigma Aldrich) were purchased to serve as positive controls for neutralization assays.
7
Antiretroviral Drug Combination Protocol
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Enfuvirtide (T-20; SML0934) was purchased from Sigma-Aldrich, Emtricitabine (FTC; ab145045) from abcam, Abacavir (ABC; Cay14746-10) from Biomol, Lamivudine (3TC; T0682) and Dolutegravir (DTG; T6198) from TargetMol, Efavirenz (EFV; 14412) and Saquinavir (SQV; 9001893) from Cayman.
8
Anti-HIV Screening of Cassia abbreviatta Extract
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MT4 cells were obtained through the NIH AIDS Reagent Program and cultured in RPMI 1640 (Lonza, Wijchen, the Netherlands) supplemented with 10% heat-inactivated fetal bovine serum (Lonza, the Netherlands) and 2mM l -glutamine (Invitrogen, Gosselies, Belgium). MT-4 cells were incubated with the crude extract of Cassia abbreviatta or the tested compounds alone to assess cytotoxicity, or HIV-1 IIIB alone or a mixture of the tested extract and compounds and HIV-1 IIIB viruses to assess protection against HIV-1 infection. After five days, protection from viral infection and the cytotoxicity were evaluated in parallel using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma, Liège, Belgium) by measuring OD540 and OD690 using a POLARstar Omega Plate Reader (BMG Labtech, Ortenberg, Germany). Data were normalized to cells without treatment. Values of OD540−OD690 were calculated to determine IC50 values in Prism. The entry inhibitors, enfuvirtide, and AMD3100 (Sigma Aldrich, Liège, Belgium), were used as positive controls.
9
Quantifying HIV-1 DNA Clearance
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Isolated CD4+ T cells obtained from HIV-1+ donors (as detailed above) were infected with HIV-WT or HIV-ΔNef. Cells were stained with the dye CellTrace Far Red (Thermo Fisher Scientific) at 100 nM (HIV-ΔNef) and 1 μM (HIV-WT) to identify each population of infected cells by flow cytometry. After 24 hours of infection, antiretrovirals were added to avoid HIV-1 spread and reinfection (100 nM Efavirenz, 100 ng/ml Enfuvirtide, MilliporeSigma). On day 3 after infection, infected cells were mixed and cocultured with autologous expanded CD8+ T cells at a 1:3 to 1:20 CD4/CD8 ratio. After 24 hours, one-half of the culture was used to measure p24 expression by flow cytometry. In parallel, the second half was washed with PBS and then incubated with 60 U DNase I for 30 minutes at 37°C to degrade the HIV-1 proviral DNA from dead cells. Next, cells were washed 3 times with PBS before performing cell lysis and FLIPS.
10
Enrichment of Activated CD4+ T Cells
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PBMCs were thawed and cultured in cRF10 medium supplemented with 100 U/ml IL-2, 0.5 μg CD3/8 bispecific antibody (ARP-12277, obtained from Johnson Wong and Galit Alter through the NIH AIDS Reagent Program), and antiretrovirals (100 nM Efavirenz, 100 ng/ml Enfuvirtide and 30 mM Raltegravir, MilliporeSigma) to avoid HIV-1 spread and reinfection. PBMC treatment with CD3/8 bispecific antibody resulted in the elimination of CD8+ T cells and enrichment of activated CD4+ T cells. Purification of CD4+ T cells was performed by negative selection using magnetic beads (Human CD4+ T cell Isolation Kit, Miltenyi Biotec).
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