Wild-type and gp41 mutant HIV-1NL4.3 virus strains (50,000, 25,000 and 12,500 pg p24) were incubated with 106 Raji/DC-SIGN cells for 1 h at 37°C in a volume of 1 ml, after which unbound virions were removed by thorough washings. The cells were resuspended in 1 ml of fresh culture medium and the amount of captured virions was determined using a p24 ELISA (PerkinElmer, Boston, MA). In parallel, 2x105 virus-captured Raji/DC-SIGN cells were brought into contact with 2x105 C8166 cells in a volume of 1 ml, resulting in the transmission of virions from the Raji/DC-SIGN cells to the C8166 cells and the subsequent infection of the C8166 cells. After 72 h of cocultivation, the formation of syncytia was evaluated microscopically and the amount of virus produced by the C8166 cells was determined by a p24 ELISA (PerkinElmer). The measured viral load was used as a parameter for the efficiency of transmission of virus from Raji/DC-SIGN cells to C8166 cells.
P24 elisa
Manufactured by PerkinElmer
Sourced in United States
The P24 ELISA is a laboratory equipment product designed for the quantitative determination of the p24 protein, a core antigen for HIV. The product utilizes the enzyme-linked immunosorbent assay (ELISA) technique to detect and measure the presence of the p24 antigen in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Steadyglo reagent, by Promega (3 mentions)
Fugene hd, by Promega (3 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Turbo dnase, by Thermo Fisher Scientific (2 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (2 mentions)
293t 17 cells, by American Type Culture Collection (2 mentions)
Hiv 1 p24 elisa kit, by PerkinElmer (1 mentions)
Kc 57rd1 pe, by Beckman Coulter (1 mentions)
Lipofectamine plus, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Hela cell, by American Type Culture Collection (1 mentions)
Polybrene, by Merck Group (1 mentions)
Clone kc57 pe, by Beckman Coulter (1 mentions)
Heparinase, by Merck Group (1 mentions)
Mtt cell viability assay kit, by Biotium (1 mentions)
Keratinocyte growth medium, by Lonza (1 mentions)
Quikchange 2 xl site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Tenofovir, by Selleck Chemicals (1 mentions)
Raltegravir, by Selleck Chemicals (1 mentions)
35 protocols using p24 elisa
1
HIV-1 Transmission Assay with Raji/DC-SIGN Cells
2
HIV Neutralization Assay with TZM-bl Cells
3
Viral Particle Isolation and Characterization
4
Quantifying Jurkat Cell HIV Infection
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For untransfected Jurkat cells, HIV infection conditions were the same as for the M7-Lue cell line, but HIV infectivity was measured by HIV-1 p24 production at 7 days post infection using the HIV-1p24 ELISA kit (PerkinElmer). For CD44− or empty vector-transfected Jurkat cells, after 3 days of transfection, the cells were infected with 5 ng of HIV-p24 for 5 h, then washed three times with complete RPMI, resuspended in complete RPMI and cultured for 3 days. These cells were analyzed for HIV infection by two methods: first, by intracellular staining for HIV-p24 (KC-57RD1-PE; Beckman Coulter, Fullerton, CA, USA) and CD44 (anti-huCD44-APC; R&D system); and second, measuring HIV-1 p24 in the supernatant by p24 ELISA (PerkinElmer).
5
Lentiviral Particle Production in HEK293T Cells
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Human embryonic kidney (HEK) 293T cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) medium with 10% FBS (Gibco) and maintained in 5% CO2 incubator at 37 °C. The lentivirus particles were produced by co-transfection of HEK 293T cells with three plasmids: VSV-G, gag-pol, and pLentiM1.9-SOD3, using Lipofectamine Plus (Invitrogen, Carlsbad, CA, USA). At 48 h post-transfection, culture supernatants containing virus particles were collected and clarified with a 0.45 μm membrane filter (Nalgene, Rochester, NY, USA) and stored at −70 °C, in a deep-freezer, immediately. Titers were determined by p24 ELISA (Perkin-Elmer Life Science, Waltham, MA, USA) or Western blot analysis, using a monoclonal anti-p24 antibody (NIH, Bethesda, MD, USA). In our routine preparation, the titers were approximately 1.0 × 107 TU/mL.
6
LV Viral Titer Quantification in HeLa Cells
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For LV-GFP flow cytometry11 (link), 27 (link) and for LV-TK, qPCR-based methods were used for analysis of the infective titers in HeLa cells (ATCC). Two HeLa cell lines grown in separate laboratories were used when the effect of titering method on the viral titers was tested. In qPCR tittering, the following primers and probe were used: WPRE Forw 5′-GGCACTGACAATTCCGTGGT-3′; WPRE Rev 5′-AGGGACGTAGCAGAAGGACG-3′ and WPRE Probe FAM 5′-CGTCCTTTCCATGGCTGCTCGC-OQA-3′ (Merck). For normalization Taqman Copy Number Reference Assay, human, RNaseP (Thermo Fischer/Life Technologies Bleiswijk, Netherlands) was used. Viral particle titer was determined by measuring p24 concentration (pg/mL) using p24 ELISA (PerkinElmer Life Sciences) and converting the results to vp/mL by assuming 12,500 LV particles per 1 pg of p24.28 , 29
7
Quantifying HIV-1 Transmission via DC-SIGN
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Raji/DC-SIGN cells (2 x 105) were exposed to WT or mutant HIV-1NL4.3 particles (equivalents of 5 ng p24) during 1h at 37°C. After thoroughly washing to eliminate unbound virions, the (virus-bound) cells were resuspended in 200μl of fresh culture medium. Next, the virus-bound Raji/DC-SIGN cells were exposed to 2 x 105 C8166 cells in a total volume of 1 ml. Following an incubation of 72h, samples were harvested to quantify the amount of HIV-1 p24 antigen using a p24 ELISA (PerkinElmer). These values were used as a measure of viral production resulting from transmission of virions from virus-captured Raji/DC-SIGN cells to CD4+ C8166 cells.
8
Pseudotyped HIV-1 Infectivity Assay
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The antiviral activity
of all RNA-targeted compounds was measured by single-round infectivity
assay with pseudotyped HIV-1 using HEK293T producer cells. The HIV-1
proviral vector (pDHIV3-GFP) includes all HIV-1 NL4–3 genes
except nef (replaced with GFP) and env, thus preserving gag and pol,
and the frameshift required for production of the Gag-Pol polyprotein.
A single-round infectivity assay was conducted by transient transfection
of the viral vector with VSV-G coat protein vector at a ratio of 1:0.5
using Fugene HD (Promega). The virus producer cells were dosed with
compounds 4 h after transfection, and viral particles were harvested
from the media 24 h after transfecting by filtering through a 0.45-μm
syringe filter. Viral load was normalized with a p24 ELISA (PerkinElmer).
The infections were performed using TZM-bl reporter cells containing
stably integrated firefly luciferase, the expression of which is driven
by the HIV-LTR promoter. Therefore, luciferase is expressed upon successful
HIV infection. Triplicate infections in 96-well plates at 10 000
cells/well with 500 pg p24/well proceeded for 48 h before the addition
of SteadyGlo reagent (Promega) to each well for 30 min. Luminescence
was measured as a quantitative metric for changes in viral infectivity
in the presence of a compound.
of all RNA-targeted compounds was measured by single-round infectivity
assay with pseudotyped HIV-1 using HEK293T producer cells. The HIV-1
proviral vector (pDHIV3-GFP) includes all HIV-1 NL4–3 genes
except nef (replaced with GFP) and env, thus preserving gag and pol,
and the frameshift required for production of the Gag-Pol polyprotein.
A single-round infectivity assay was conducted by transient transfection
of the viral vector with VSV-G coat protein vector at a ratio of 1:0.5
using Fugene HD (Promega). The virus producer cells were dosed with
compounds 4 h after transfection, and viral particles were harvested
from the media 24 h after transfecting by filtering through a 0.45-μm
syringe filter. Viral load was normalized with a p24 ELISA (PerkinElmer).
The infections were performed using TZM-bl reporter cells containing
stably integrated firefly luciferase, the expression of which is driven
by the HIV-LTR promoter. Therefore, luciferase is expressed upon successful
HIV infection. Triplicate infections in 96-well plates at 10 000
cells/well with 500 pg p24/well proceeded for 48 h before the addition
of SteadyGlo reagent (Promega) to each well for 30 min. Luminescence
was measured as a quantitative metric for changes in viral infectivity
in the presence of a compound.
9
Measuring Antiviral Activity of Compounds
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The antiviral activity of 4 , 5 , and 10 was measured by single-round
infectivity assay with pseudotyped HIV-1 using HEK293T producer cells.
The HIV-1 proviral vector (pDHIV3-GFP) codes for all HIV-1NL4–3 genes except nef (replaced with GFP) and env, thus preserving gag and pol, and the frameshift required for production of the Gag-Pol polyprotein.
A single-round infectivity assay was conducted by transient transfection
of the viral vector with VSV-G coat protein vector at a ratio of 1:0.5
using Fugene HD (Promega). The virus producer cells were dosed with
compounds four hours after transfection, and viral particles were
harvested from the media 24 h after transfecting by filtering through
a 0.45-μm syringe filter. Viral load was normalized with a p24
ELISA (Perkin-Elmer).
The infections were performed using TZM-bl
reporter cells that contain stably integrated firefly luciferase that
is driven by the HIV-LTR promoter. Therefore, luciferase is expressed
upon successful HIV infection.53 (link) Triplicate
infections in 96-well plates at 10,000 cells/well with 500 pg p24/well
proceeded for 48 h before the addition of SteadyGlo Reagent (Promega)
to each well for 30 min. Luminescence was measured as a quantitative
metric for changes in viral infectivity in the presence of compound.
infectivity assay with pseudotyped HIV-1 using HEK293T producer cells.
The HIV-1 proviral vector (pDHIV3-GFP) codes for all HIV-1NL4–3 genes except nef (replaced with GFP) and env, thus preserving gag and pol, and the frameshift required for production of the Gag-Pol polyprotein.
A single-round infectivity assay was conducted by transient transfection
of the viral vector with VSV-G coat protein vector at a ratio of 1:0.5
using Fugene HD (Promega). The virus producer cells were dosed with
compounds four hours after transfection, and viral particles were
harvested from the media 24 h after transfecting by filtering through
a 0.45-μm syringe filter. Viral load was normalized with a p24
ELISA (Perkin-Elmer).
The infections were performed using TZM-bl
reporter cells that contain stably integrated firefly luciferase that
is driven by the HIV-LTR promoter. Therefore, luciferase is expressed
upon successful HIV infection.53 (link) Triplicate
infections in 96-well plates at 10,000 cells/well with 500 pg p24/well
proceeded for 48 h before the addition of SteadyGlo Reagent (Promega)
to each well for 30 min. Luminescence was measured as a quantitative
metric for changes in viral infectivity in the presence of compound.
10
Lentivirus-mediated ERFE Overexpression
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Lentivirus-based vectors pRRL-sin-cPPT-hCMV-MCS-IRES-emdGFP encoding ERFE mouse cDNA ORF (accession number NM_173395) fused to a C-terminal 3xFLAG tag and empty control vector were produced by the UCLA Vector Core facility. Internal ribosome entry site ensured reinitiation and translation of Green Fluorescent Protein (GFP). Vector titers were determined by p24 ELISA (Perkin Elmer). HEK293T cells (ATCC® CRL-3216™) were transduced using 4 μg/ml Polybrene (Sigma, H9268). Insertion of the virus was confirmed by GFP detection by fluorescence microscopy. ERFE overexpression and secretion was monitored by western blot.
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