Mice were infected with a luciferase-expressing strain of P. chabaudi (PccASluc [45 (link)]) as described above but kept on a reverse light cycle, as sequestration occurs during the dark cycle [45 (link)]. At the time of maximum sequestration (12.00 – 14.00 h coordinated universal time (UCT), reverse light) mice were sacrificed and perfused systemically by injection of 10 ml PBS into the heart. Organs were harvested and 0.1 g of tissue was transferred to a Precellys homogenizer tube in PBS and dissociated for one cycle, 10 seconds at 4500 rpm in a Precellys Evolution Homogenizer. The sample was then diluted 1:10 in PBS, and an equal volume (100 μL) of Bright-Glo substrate (Promega) was added. Luciferase activity was measured after 2 min incubation using a Perkin Elmer VICTOR X Light Multilabel Plate Reader.
Bright glo substrate
Manufactured by Promega
Sourced in United States
Bright-Glo is a luminescent substrate designed to be used in luciferase reporter assays. It is optimized to provide a bright, stable luminescent signal for the quantitative measurement of luciferase activity.
Automatically generated - may contain errors
Lab products found in correlation
Victor x5, by PerkinElmer (3 mentions)
Bright glo lysis buffer, by Promega (2 mentions)
Victor x light multilabel plate reader, by PerkinElmer (1 mentions)
Glomax navigator, by Promega (1 mentions)
Attractene transfection reagent, by Qiagen (1 mentions)
Blasticidin, by InvivoGen (1 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
Glomax 96 microplate luminometer, by Promega (1 mentions)
Glomax luminometer, by Promega (1 mentions)
Synergy mx luminometer, by Agilent Technologies (1 mentions)
Vacutainer serum collection tube, by BD (1 mentions)
Pap1 pma ta luc, by Takara Bio (1 mentions)
Pgl4.33 sre, by Promega (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
Nunclon delta surface, by Thermo Fisher Scientific (1 mentions)
Dual luciferase assay system, by Promega (1 mentions)
2030 manager software, by PerkinElmer (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Glomax navigator luminometer, by Promega (1 mentions)
Glomax discover system, by Promega (1 mentions)
18 protocols using bright glo substrate
1
Quantifying Parasite Sequestration In Mice
2
BCMA-CAR T Cell Activation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The JNL reporter cell line was engineered from Jurkat E-6 cells and contains the firefly luciferase gene under the control of a minimal (m) CMV promoter and tandem repeats of the NFAT transcriptional response element. Cells were cultured in RPMI with 10% FBS and 0.5 μg/ml puromycin. JNL cells transduced with BCMA-targeting CAR constructs were evaluated for activation in response to BCMA-expressing target cell lines. On Day 3 following transduction, transduced JNL cells were incubated with target cells in duplicate at an effector-to-target (E:T) ratio of 6:1. JNL activation was measured using Bright-Glo substrate (Promega) on Day 4.
3
Pseudotyped Particle Infectivity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For pseudotyped particles with a luciferase reporter, the target cells were seeded at 15,000 cells per well into a 96-well plate and infected with the supernatant containing pseudotyped particles, with a final concentration of 4 μg/mL of Polybrene. The plates were spin inoculated at 1,240 RCF for 30 min at 20°C. Approximately 72 h later, the cells were lysed by the addition of 100 μL of a 1:1 (vol/vol) mixture of phenol red-free DMEM and Bright-Glo substrate (Promega), and infectivity was determined as the relative luminescence units (RLU) per well using Glomax Navigator (Promega). For pseudotypes carrying a GFP reporter gene, target cells were seeded at 100,000 cells per well into a 24-well plate and transduced with the supernatant containing pseudotypes, with a final concentration of 4 μg/mL of Polybrene, and incubated at 37°C for 72 h. The cells were then detached from the plate with trypsin, fixed with 4% paraformaldehyde in PBS, and analyzed on a FACSCanto II instrument for GFP expression.
4
Octβ2R and OA1.0 transfection effects
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HTLA cells were cultured in 6-well plates; at ~70% cell density, the cells were transfected with either wild-type Octβ2R or OA1.0. Twenty-four hours after transfection, the cells were transferred to a 96-well white clear flat-bottom plate, and virous concentrations of OA (ranging from 1 nM to 100 μM) were added to the cells; each concentration was applied in triplicate. The cells were then incubated for ~16 hours, and the bioluminescent signal was measured. To measure the bioluminescent signal, the culture medium was removed, and 40 μl of Bright-Glo substrate (Promega) was added to the wells. The plate was then incubated at room temperature in the dark for 10 minutes, and the bioluminescent signal was measured using a Victor X5 microplate reader (PerkinElmer). Non-transfected cells were used as negative controls.
5
Quantifying NF-κB Activity in hTLR2 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293 cells overexpressing human TLR2 (293-hTLR2; In vivoGen, San Diego, CA, USA) were maintained in Dulbecco’s modified eagle medium containing 10% FBS, 1% penicillin-streptomycin solution, and 10 μg/ml blasticidin (In vivoGen). The 293-hTLR2 cells (5 × 105 cells/ml) were transfected with NF-κB luciferase reporter construct (Clontech Laboratories, Mountain View, CA, USA) using Attractene transfection reagent (QIAGEN®, Venlo, Netherlands). After stimulating the transfected cells with either 1 × 107 and 1 × 108 CFU/ml of wild-type or ΔltaS HKSG for 20 h, the cell lysate was prepared with Passive Lysis Buffer (Promega, Madison, WI, USA). Then, Bright-Glo™ substrate (Promega) was added to the cell lysate to quantify the luciferase activity with GloMax® 96 Microplate Luminometer (Promega).
6
Pseudovirus Infection Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Target cells were seeded into white 96-well plates 24 h before infection (Caco2 and Vero.E6 cells seeded at 1.5 × 104 cells/well, and HeLa-TZMbl cells seeded at 1 × 104 cells/well). Cells were infected with increasing doses (2 to 15 mU RT/well) of PV supernatant (to confirm linear increase in infection at increased PV doses) and incubated at 37 °C for 48 h without changing the medium. Luciferase expression (RLU) was measured at 48 h postinfection using BrightGlo substrate (Promega) according to the manufacturer’s instruction on the Glomax luminometer (Promega). For inhibitor studies, cells were pretreated before infections for 2 h with Camostat mesylate (Apexbio, 0.2 to 100 μM) or E64d (Focus Biomolecules, 0.2 to 25 μM). To obtain infectivity (RLU/RT) values, RLU values were normalized to input supernatant RT activity (measured by SG-PERT assay, described above).
7
Neutralizing Antibody Screening Assay for AAV2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole blood was obtained in Vacutainer Serum Collection Tubes (Becton Dickinson) and serum was collected following centrifugation at 1500 rcf/g for 10 min and stored at −80C until analysis. Neutralizing antibody screening assays were carried out as previously described53 (link). Briefly, assays were performed in 96 well format with 5 × 104 CHO-Lec2 cells per well. Serial dilutions of study participant serum were pre-incubated with 1 × 109 genome copies of AAV2 reporter virus for 1 hour at 37 °C and then added to cells that were infected with Adeno Helper virus. After 48 hours, Promega Bright-Glo substrate was added to the cells and luciferase expression was quantified using the Biotek Synergy Mx luminometer. Both positive and negative monkey sera controls were included with each assay. All animals selected for the study had less than 50% inhibition of transduction when serum was diluted to 1:20.
8
Virus Neutralization Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibody and inhibitor neutralisation assay was performed as described previously [40 (link)]. Briefly, virus supernatants were incubated with serial dilutions of soluble CD4 (CFAR NIBSC), AMD3100 (CFAR, NIBSC), PGT151 [41 (link)] (gift from Laura McCoy, UCL) or T-20 fusion inhibitor (CFAR, NIBSC) and incubated at 37 °C for 1 h. The mixture was added to TZM-bl cells, and luciferase activity measured 48 h later using Bright-Glo substrate (Promega, Madison, WI, USA). Neutralisation was calculated as percent decrease in luciferase activity compared to virus-only control. IC50 values were calculated by nonlinear regression analysis (sigmoid curve interpolation) using Prism software (GraphPad, San Diego, CA, USA).
9
Luciferase Reporter Assay for AP-1 and SRE
10
SARS-CoV-2 Pseudotyped Lentivirus Infection Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lentiviruses pseudotyped with the SARS-CoV-2 spike were generated in HEK293T cells as described previously [28 (link),29 (link)]. Briefly, HEK293T cells were co-transfected with a SIV-based self-inactivating vector encoding luciferase (pGAE-LucW), the SIV-based packaging plasmid (pAdSIV3), and a WT spike-encoding plasmid. Pseudoviruses were harvested 72 h after transfection and used for the infection of 293T-DSP-mix cells transfected with an ACE2 expression construct or empty vector (mock control). At 48 hpi, cells were lysed through the addition of 100 µL Bright Glo lysis buffer (Promega) for 15 min at 37 °C. Three minutes later, after the addition of 25 µL Bright Glo substrate (Promega), the luciferase signals were measured using a microplate luminometer (VICTOR X5, PerkinElmer) and the PerkingElmer 2030 Manager software.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!