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Victor light 2030 luminometer

Manufactured by PerkinElmer

The Victor Light 2030 luminometer is a sensitive and versatile instrument designed for a wide range of luminescence-based assays. It measures light emission from samples in microplates, enabling quantitative analysis of various biological and chemical processes. The Victor Light 2030 offers reliable performance and flexible configuration options to support diverse laboratory applications.

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5 protocols using victor light 2030 luminometer

1

HIV-1 Pseudovirus Neutralization Assay

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Two HIV-1 pseudoviruses, tier 1 and tier 2 virus strains, respectively with a higher and lower level of neutralization susceptibility were employed in the TZM-bl assay: SF162 (Clade B, Tier 1 virus), QH0692 (Clade B, Tier 2). Viral stocks were generated and titered. In particular, 293 T cell-derived stocks of pseudoviruses were generated by proviral DNA transfection using FuGENE HD, according to the manufacturer’s protocol (Promega, Madison, WI). Viral supernatants were harvested 72 h post transfection and clarified at 1800 rpm for 20 min. The virus stocks were analysed for firefly luciferase expression in the TZM-bl cell line. Four replicates of five-fold dilutions of viruses were added to 96 flat-bottomed plate wells containing 104 TZM-bl cells per well, in DMEM supplemented with 10% FBS and 7.5 μg/ml DEAE-dextran in a final volume of 200 μl. After 48 h of incubation at 37°C, 100 μL of culture medium were removed from each well and replaced with 100 μL of Bright Glo luciferase reagent (Promega). After 2 min of incubation, 100 μL of the cell lysate were transferred to a 96 well white solid plate and luminescence was measured using a Victor Light 2030 luminometer (Perkin Elmer). Neutralization titers were expressed as ID50 values, defined as the virus dilution required to achieve half maximal infection (Reed–Muench calculation).
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2

Pseudovirus Production and Titration

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293 T cell-derived stocks of pseudoviruses were generated by proviral DNA transfection using FuGENE 6, according to the manufacturer's protocol (Promega, Madison, WI). Viral supernatants were harvested 72 h post transfection, clarified at 1800 rpm for 20 min, and frozen at –70 °C. The virus stocks were further analyzed for firefly luciferase expression in the TZM-bl cell line. Four replicates of five-fold dilutions of viruses were added to 96 flat-bottomed plate wells containing 104 TZM-bl cells per well, in 10% D-MEM growth medium with 7.5 μg ml–1 DEAE-dextran (Sigma) in a final volume of 200 μl. After 48 h of incubation at 37 °C, 100 μL of culture medium was removed from each well and replaced with 100 μL of Bright-Glo luciferase reagent (Promega). After 2 min of incubation, 150 μL of the cell lysate was transferred to a 96-well white solid plate and luminescence was measured using a Victor Light 2030 luminometer (Perkin Elmer). Fifty percent infectious dose (ID50) titers were defined as the reciprocal of the virus dilution yielding 50% positive wells (Reed–Muench calculation).
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3

Pseudovirus Infectivity Quantification Protocol

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293 T cell-derived stocks of pseudoviruses and replication-competent IMCs were generated by proviral DNA transfection using FuGENE 6, according to the manufacturer’s protocol (Promega, Madison, WI). Viral supernatants were harvested 72 h post-transfection, clarified at 1800 rpm for 20 min, and frozen at −70°C. The virus stocks were further analyzed for firefly luciferase expression in the TZM-bl cell line. Four replicates of five-fold dilutions of virus were added to 96 flat-bottomed plate wells containing 1 × 104 TZM-bl cells per well, in 10% D-MEM growth medium with 7.5ug/ml of DEAE-dextran (Sigma) in a final volume of 200ul. After 48 h incubation at 37°C, 100uL of culture medium were removed from each well and replaced with 100 uL of Bright-Glo luciferase reagent (Promega). After 2 min incubation, 150 uL of the cell lysate was transferred to a 96-well white solid plate and luminescence was measured using a Victor Light 2030 luminometer (Perkin Elmer). Fifty percent infectious dose (ID50) titers were defined as the reciprocal of the virus dilution yielding 50% positive wells (Reed-Muench calculation).
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4

Quantifying Intracellular Leishmania in BMM

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Intracellular amastigotes in BMM from Ctsb−/− and Ctsl−/− mice, and their WT C57BL/6 counterparts, were measured with the method described by Bringmann et al. [30] (link). Briefly, 200 µl of a 2×105 cells/ml suspension of BMM in phenol red-free complete medium were seeded into 96-well plates with clear bottoms (Greiner Bio-One, Frickenhausen, Germany) and were incubated for 4 hours to allow cell adhesion. The medium was then removed, and 200 µl of a 3×106 cells/ml suspension of Luc-tg L. major promastigotes were added at an infection ratio of 1∶15 and incubated for 24 hours at 37°C, 5% CO2. Any remaining extracellular parasites were eliminated by washing 3 times with medium, and 200 µl of the phenol red medium were added. After further 24 hours incubation at 37°C, 5% CO2, 50 µl of the luciferin-containing lysis buffer Britelite Plus (PerkinElmer, Waltham, USA) were added to each well. The plate was incubated in the dark for 5 min at room temperature (RT), and the resulting luminescence was measured as counts per second (CPS), with a Victor×Light 2030 luminometer (PerkinElmer).
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5

Antibody Neutralization Assay for Pseudoviruses

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Six 3-fold serial dilutions of antibodies samples (starting from 66ug/mL), were plated in triplicate (96-well flat bottom plate) in 10% D-MEM growth medium (100 uL/well). 200 TCID50 of each pseudovirus or 20 TCID50 of each T/F IMC were added to each well in a volume of 100 uL and incubated for 1 h at 37°C. TZM-bl cells were then added (1 × 104/well in a 100 uL volume) in 10% D-MEM growth medium containing DEAE-dextran (Sigma), at a final concentration of 7.5 ug/mL. Assay controls included replicate wells of TZM-bl cells alone (cell control) and TZM-bl cells with virus (virus control). Following a 48 h incubation at 37°C, 150 uL of culture medium were removed from each well and replaced with 100 uL of Bright-Glo luciferase reagent (Promega). After a 2-min incubation, 150 uL of the cell lysate was transferred to a 96-well black solid plate and luminescence was measured using a Victor Light 2030 luminometer (Perkin Elmer). The 50% inhibitory dose (IC50) was calculated as the concentration of antibody that induced a 50% reduction in relative luminescence units (RLU) compared to the virus control wells, after subtraction of cell control RLU.
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