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39 protocols using victor 3 luminometer

1

Neutralization Titers of Monoclonal Antibodies

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Neutralization titers of monoclonal Abs were determined using a luciferase-based assay in TZM.bl cells as previously described.50 (link), 51 (link) Briefly, mAb samples were tested using a primary concentration of 25 μg/mL with 5-fold serial dilutions against a panel of 10 HIV-1 Env pseudoviruses that were selected for being either 3BNC117 sensitive/PGT121 resistant or 3BNC117 resistant/PGT121 sensitive. This allowed for the measurement of neutralizing activity of single Abs in samples containing a mixture of both 3BNC117 and PGT121. Antibody titrations were incubated with HIV-1 Env pseudoviruses for 1 h at 37°C, and TZM.bl cells were then added in growth media containing DEAE-dextran at a final concentration of 11 μg/mL. Assay plates were incubated for 48 h at 37°C, 5% CO2, and luciferase reporter gene expression was measured using Bright-Glo luciferase reagent (Promega) and a Victor 3 luminometer (Perkin Elmer). Neutralization titers (50% and 80% inhibitory concentrations, IC50 and IC80, respectively) were calculated as the mAb concentration at which relative luciferase units (RLU) were reduced by 50% or 80% compared to RLU in virus control wells after subtraction of background RLU in cell control wells. All assays were performed in a laboratory meeting good current laboratory practice standards.
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Proteasomal Activity Profiling in DMD Myoblasts

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The control and DMD myoblasts cell lines were plated in 96-well culture plates at a density of 104 cells/well. The day after transfection, proteasome activities (caspase-, trypsin- and chymotrypsin-like activities) were measured using cell-based Proteasome-Glo™ assay (Promega) as previously described [42 (link)]. The proteasome inhibitors MG132 and lactacystin (10 µM, 2 h) were used as negative controls. Luminescence was measured using a Victor3 Luminometer (Perkin Elmer). The relative light units produced were reported to 50 µg of total proteins.
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Assessing TIGK Cell Metabolic Activity

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The metabolic activity of TIGK cells was assessed by measuring total ATP levels using the CellTiter-Glo reagent (Promega, Madison WI), as described by the manufacturer. TIGK cells were seeded at a density of 6 × 104 cells in 1 mL media per well and incubated at 37°C, 5% CO2 for 24 h in a 12-well flat bottom plate. Cells were then incubated with BAR or 10:90 PLGA-PEO BAR-EFs (1.3 or 3.4 μM) for 24 h at 37°C in 5% CO2. Cells were then lysed with 500 μL of 0.1% Triton X-100 for 30 min at 37°C. The lysates were collected and centrifuged at 1,000 × g for 10 min at 4°C, and 50 μL of supernatant was mixed with 50 μL of CellTiter-Glo reagent. Samples were incubated at ambient temperature for 10 min in a black 96-well plate in the dark. Total luminescence was measured with a Victor 3 luminometer (Perkin-Elmer, Inc.). Cells incubated with 1 ng of staurosporine or with medium-only served as positive and negative controls for cell death, respectively (Mahmoud et al., 2019 (link)).
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Transcription Factor Activation in HUVECs

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HUVECs were treated with proHp CM (final proHp: 0.2 μg/ml), and incubated for 14 h. Nuclear proteins from the cells were extracted with the Nuclear Extraction Kit (Signosis, Santa Clara, CA, USA), and transcription factor activation was detected using the Transcription Factor Activation Array І (Signosis), according to the manufacturer’s instructions. Briefly, the nuclear extracts were incubated with the probe mix for 30 min at room temperature, and then the transcription factor/probe complexes were bound to a spin column. The bound probes were separated from the complexes with elution buffer and hybridized to plates precoated with DNA sequences that are complementary to the specific probes. The captured probes were detected with streptavidin-HRP. The luminescence was measured on a Victor3 luminometer (PerkinElmer, Boston, MA, USA) and expressed as relative light units (RLUs).
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5

Stable 9464D Cell Line with GFP-Luciferase

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9464D cells were transfected with a construct containing a green fluorescent protein (GFP)-Ires-Luciferase sequence under the control of a cytomegalovirus (CMV) promoter. Stably transfected 9464D cells were selected by adding increasing amounts of G418 to the medium to a final concentration of 200 ng/ml. GFP-positive cells were FACsorted and plated as single cells in 96-well plates. GFP-positive clones were expanded and selected for stable transfection by monitoring GFP-positivity by FACS. Luciferase expression was determined using a luciferase reporter assay; 1 × 105 cells were lysed in 100 μl Passive Lysis Buffer (Promega), and the lysates were analyzed for luminescence with the use of the Dual-luciferase Reporter Assay System (Promega, Leiden, the Netherlands) according to manufacturer’s protocol and a Victor 3 luminometer (PerkinElmer, Groningen, the Netherlands). Several stably transfected clones were expanded, tested for cell surface marker expression and frozen for later use.
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Cytotoxicity Evaluation of PEI-Coated Nanoparticles

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The in vitro cellular cytotoxicities of PEI-coated nanoparticles were measured using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA). The intracellular adenosine triphosphate was quantified using a Victor 3 luminometer (Perkin Elmer, Waltham, MA, USA). The human prostate cancer (DU145) cell line (Korean Cell Line Bank, Seoul, Korea) was used. The RPMI1640 was used as a cell culture medium. The cells were seeded on a separate 24-well cell culture plate and incubated for 24 h. Five test sample solutions (10, 50, 100, 200, and 500 μM Ho) were prepared by diluting the concentrated original nanoparticle suspension samples with a sterile phosphate-buffered saline solution and 2 mL aliquots were used to treat the cells, which were subsequently incubated for 48 h. Cell viabilities were measured thrice to obtain the average cell viabilities, which were then normalized in terms of the viability of untreated control cells (0.0 mM Ho).
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7

SARS-CoV-2 Spike Pseudotyped Lentivirus Assay

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A SARS-CoV-2 spike pseudotyped lentivirus kit was obtained through BEI Resources, NIAID, NIH (SARS-Related Coronavirus 2, Wuhan-Hu-1 Spike-Pseudotyped Lentiviral Kit V2, NR-53816). We used HEK-293T–expressing human angiotensin-converting enzyme 2, also known as HEK-293T–hACE2, which are susceptible to SARS-CoV-2. This cell line was obtained through BEI Resources, NIAID, NIH, NR-52511. Serial dilutions of sera were incubated with the SARS-CoV-2 spike pseudotyped lentivirus, following a protocol by Balazs and Bloom (50 ). Cells were lysed using luciferase cell culture lysis buffer (Promega). Luciferase reaction was performed using 30 μl of cell lysis (Promega). The reaction was added to 96-well black optiplates (PerkinElmer). Luminescence was measured using a PerkinElmer Victor3 luminometer.
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8

Cytotoxicity Evaluation of Gadolinium Nanoparticles

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The in vitro cellular cytotoxicity of the aqueous nanoparticle suspension samples was measured using a CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA). The intracellular adenosine triphosphate was quantified using a Victor 3 luminometer (Perkin Elmer, Waltham, MA, USA). Two cell lines, NCTC1469 and DU145, were used. Each cell line was seeded onto a separate 24-well cell culture plate and incubated for 24 h (5 × 104 cell density, 500 μL cells/well, 5% CO2, and 37 °C). Nine test solutions (0.01, 0.05, 0.1, 0.2, 0.5, 1, 2, 3, 5 mM Gd) were prepared by diluting the original concentrated nanoparticle suspension sample dispersed in triple-distilled water with a sterile phosphate-buffered saline (PBS) solution. Afterward, 2 μL aliquots were used to treat the cells, which were subsequently incubated for 48 h. Cell viabilities were measured thrice to obtain average cell viabilities, which were normalized with respect to that of the untreated control cells (0.0 mM Gd).
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9

Neutralizing Antibody Assay for HIV

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Patient plasma samples were heat-inactivated (56°C for 1 hour) and tested for neutralizing activity using a luciferase-based assay in TZM.bl cells as previously described [35 (link)]. Briefly, three-fold dilutions of plasma samples starting at a 1:20 dilution were performed in duplicate. Env-pseudotyped viruses were added to the plasma dilutions at a pre-determined titre to produce measurable infection and incubated for one hour at 37°C. TZM.bl cells were then added at 1×104/well and plates incubated for 48 hours. Cells were lysed for two minutes with Bright-Glo luciferase reagent (Promega, Madison, WI, USA), and supernatant measured for luciferase activity using a Victor 3 luminometer (Perkin Elmer, Waltham, MA, USA). The 50% inhibitory dose (ID50) was calculated as the plasma dilution that resulted in a 50% reduction in relative luminescence units compared with virus control wells. All plasma samples were assayed against a standard reference panel of 11 clade B Tier 2/3 Env pseudoviruses [36 (link)]. Plasma neutralizing activity against each HIV Env pseudovirus was scored as positive, when ID50 titres were at least three-fold above Murine Leukaemia Virus negative control as previously described [37 ], and is summarized in Supplementary file 2.
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10

Validating Target Gene Regulation by miRNA

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For target gene validation, we constructed a reporter vector containing the 3′UTR of the target gene. A luciferase assay was performed using the Dual-GLO Luciferase Assay System (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Briefly, we seeded human embryonic kidney (HEK-293) cells into 12-well plates, and cotransfected the cells with 1.5 μg of pmirGLO plasmid and 20 nM or 50 nM miRNA mimics. After further cultivation for 72 h, cells were harvested and the luciferase activities were measured using a Victor3 luminometer (PerkinElmer Life and Analytical Sciences, Waltham, MA, USA). Relative luciferase activities were calculated as the ratio of firefly to Renilla luciferase activity. For measuring NFAT and MEF2 transcription factor activity, luciferase assays were performed as previously described with minor modifications (16 (link)). Briefly, the 9×NFAT- or 3×MEF2-luciferase reporter plasmid with pRL-TK containing the Renilla luciferase gene was cotransfected into NRVMs with 20 nM miRNA mimics. After 24 h, to induce hypertrophy, cells were stimulated with 10 nM ET-1 for 24 h. The luciferase reporter plasmid driven by 9×NFAT binding sites was kindly provided by Dr. Jeffery D. Molkentin (University of Cincinnati).
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