To assess cell-to-cell fusion, 2 × 106 293T cells were co-transfected with plasmid expressing HIV-1 Tat (1μg) and a plasmid expressing SARS-CoV-2 Spike (4 μg) using the calcium phosphate method. Two days after transfection, Spike-expressing 293T (effector cells) were detached with PBS-EDTA 1mM and incubated for 1 hour with indicated amounts of CV3-1 and/or CV3-25 NAbs at 37°C and 5% CO2. Subsequently, effector cells (1 × 104) were added to TZM-bl-ACE2 target cells that were seeded at a density of 1 × 104 cells/well in 96-well luminometer-compatible tissue culture plates 24 h before the assay. Cells were co-incubated for 6 h at 37°C and 5% CO2, after which they were lysed by the addition of 40 μL of passive lysis buffer (Promega) and one freeze-thaw cycles. An LB 941 TriStar luminometer (Berthold Technologies) was used to measure the luciferase activity of each well after the addition of 100 μL of luciferin buffer (15 mM MgSO4, 15 mM KH2PO4 [pH 7.8], 1 mM ATP, and 1 mM dithiothreitol) and 50 μL of 1 mM d-luciferin potassium salt (ThermoFisher Scientific).
D luciferin potassium salt
Manufactured by Thermo Fisher Scientific
Sourced in Germany
D-luciferin potassium salt is a laboratory reagent used in bioluminescence assays. It serves as a substrate for luciferase enzymes, enabling the detection and quantification of luciferase-expressing cells or reporter genes.
Automatically generated - may contain errors
Lab products found in correlation
Passive lysis buffer (plb), by Promega (4 mentions)
Lb942 tristar luminometer, by Berthold Technologies (3 mentions)
Tristar lb 941 luminometer, by Berthold Technologies (2 mentions)
96 well luminometer compatible tissue culture plates, by PerkinElmer (1 mentions)
Nightshade lb 985 in vivo plant imaging system, by Berthold Technologies (1 mentions)
Lysis buffer, by Promega (1 mentions)
In vivo xtreme imaging system, by Bruker (1 mentions)
Mi se software, by Bruker (1 mentions)
Nmri foxn1nu foxn1nu mice, by Janvier Labs (1 mentions)
Synergy 2 spectrophotometer, by Agilent Technologies (1 mentions)
Cycloheximide (chx), by Thermo Fisher Scientific (1 mentions)
Niclosamide, by Merck Group (1 mentions)
Tween 80, by Merck Group (1 mentions)
10 protocols using d luciferin potassium salt
1
Cell-Cell Fusion Inhibition Assay
2
SARS-CoV-2 Pseudovirus Neutralization Assay
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293T cells were transfected with the lentiviral vector pNL4.3 R-E- Luc (NIH AIDS Reagent Program) and a plasmid encoding for the indicated S glycoprotein (D614G, B.1.1.7, D614G/E484K, D614G/N501S, D614G/S477N, D614G/N501Y and SARS-CoV-1) at a ratio of 5:4. Two days post-transfection, cell supernatants were harvested and stored at −80°C until use. 293T-ACE2 target cells were seeded at a density of 1 × 104 cells/well in 96-well luminometer-compatible tissue culture plates (Perkin Elmer) 24 h before infection. Pseudoviral particles were incubated with the indicated plasma dilutions (1/50; 1/250; 1/1250; 1/6250; 1/31250) for 1 h at 37°C and were then added to the target cells followed by incubation for 48 h at 37°C. Then, cells were lysed by the addition of 30 μL of passive lysis buffer (Promega) followed by one freeze-thaw cycle. An LB942 TriStar luminometer (Berthold Technologies) was used to measure the luciferase activity of each well after the addition of 100 μL of luciferin buffer (15mM MgSO4, 15mM KPO4 [pH 7.8], 1mM ATP, and 1mM dithiothreitol) and 50 μL of 1mM d-luciferin potassium salt (Thermo Fisher Scientific). The neutralization half-maximal inhibitory dilution (ID50) represents the plasma dilution to inhibit 50% of the infection of 293T-ACE2 cells by SARS-CoV-2 pseudoviruses.
3
CmNAC34 Transcription Factor Regulation
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The CDS of CmNAC34 was amplified and ligated into the PRI101 vector to yield an effector construct, 35S::CmNAC34. The promoter region of CmLCYB was cloned into the pRI-mini35S-LUC vector to produce a reporter construct, and the empty pRI-mini35S-LUC was used as a control. All resulting constructs were transformed into Agrobacterium tumefaciens stain EHA105. Tobacco leaves were used for co-infiltration and treated with 1 mM D-Luciferin Potassium Salt (Thermo Fisher Scientific, Waltham, MA, USA) after three days. The luciferase fluorescence signal was detected using the NightShade LB 985 In Vivo Plant Imaging System (Berthold, Bad Wildbad, German), and the fluorescence image was taken using the IngiGo program.
4
SARS-CoV-2 pseudovirus entry assay
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293T cells were transfected with the lentiviral vector pNL4.3 R-E- Luc (NIH AIDS Reagent Program) and plasmid encoding for the indicated S glycoprotein (D614G, B.1.1.7, D614G/E484K, D614G/N501S, D614G/S477N and D614G/N501Y) at a ratio of 5:4. Two days post-transfection, cell supernatants were harvested and stored at −80°C until use. The RT activity was evaluated by measure of the incorporation of [methyl-3H]TTP into cDNA of a poly(rA) template in the presence of virion-associated RT and oligo(dT). Normalized amount of RT activity pseudoviral particles were added to 293T-ACE2 target cells for 48 h at 37°C. Then, cells were lysed by the addition of 30 μL of passive lysis buffer (Promega) followed by one freeze-thaw cycle. An LB942 TriStar luminometer (Berthold Technologies) was used to measure the luciferase activity of each well after the addition of 100 μL of luciferin buffer (15mM MgSO4, 15mM KPO4 [pH 7.8], 1mM ATP, and 1mM dithiothreitol) and 50 μL of 1mM d-luciferin potassium salt (Thermo Fisher Scientific). RLU values obtained were normalized to D614G.
5
Quantifying SARS-CoV-2 Spike-Mediated Cell Fusion
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To assess cell-to-cell fusion, 2 × 106 293T cells were co-transfected with plasmid expressing HIV-1 Tat (1μg) and a plasmid expressing SARS-CoV-2 Spike (4 μg) using the calcium phosphate method. Two days after transfection, Spike-expressing 293T (effector cells) were detached with PBS-EDTA 1mM and incubated for 1 h with indicated amounts of CV3-1 and/or CV3-25 NAbs at 37°C and 5% CO2. Subsequently, effector cells (1 × 104) were added to TZM-bl-ACE2 target cells that were seeded at a density of 1 × 104 cells/well in 96-well luminometer-compatible tissue culture plates 24 h before the assay. Cells were co-incubated for 6 h at 37°C and 5% CO2, after which they were lysed by the addition of 40 μL of passive lysis buffer (Promega) and one freeze-thaw cycles. An LB 941 TriStar luminometer (Berthold Technologies) was used to measure the luciferase activity of each well after the addition of 100 μL of luciferin buffer (15 mM MgSO4, 15 mM KH2PO4 [pH 7.8], 1 mM ATP, and 1 mM dithiothreitol) and 50 μL of 1 mM d-luciferin potassium salt (ThermoFisher Scientific).
6
HIV-1 Env Glycoprotein Neutralization Assay
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JRFL, CH058, CH077, YU2, BG505, ZM246F-10, STCO1, RHGA, CH040, and CH198 infectious molecular clones of HIV-1 were produced by the calcium phosphate transfection of HEK 293T cells. Two days after transfection, the cell supernatants were harvested. Twenty-four hours before infection, the Cf2Th-T4R5 cells were seeded at a density of 6 × 103 cells/well in 96-well luminometer-compatible tissue culture white plates (Perkin Elmer). Viruses at a final volume of 200 µL were incubated with the indicated temsavir or DMSO dilution (5, 2.5, 1.25, 0.625, 0.3125, 0.156, or 0.078 nM) for 1 h at 37 °C and were then added to the target cells in triplicate, followed by an incubation of 48 h at 37 °C. Cells were then lysed by the addition of 40 µL of a lysis buffer (Promega, Madison, WI, USA), followed by at least 1 h at −80 °C. An LB942 Tri-Star luminometer (Berthold Technologies, Bad Wildbad, Germany) was used to measure the luciferase activity of each well after the addition of 100 mL of a luciferin buffer (15 mM MgSO4, 15 mM KPO4 (pH 7.8), 1 mM ATP, and 1 mM dithiothreitol) and 50 mL of 1 mM d-luciferin potassium salt (ThermoFisher Scientific). The neutralization half-maximal inhibitory dilution (IC50) represented the temsavir dilution to inhibit 50% of the infection of Cf2Th-T4R5 cells by viruses bearing the indicated surface glycoproteins.
7
Evaluation of Immunotherapy for Prostate Cancer
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The animal experiment was performed with 8-week-old male NMRI-Foxn1nu/Foxn1nu mice (Janvier Labs, St. Berthevin, France) at the Helmholtz-Zentrum Dresden-Rossendorf according to the guidelines of German Regulations of Animal Welfare and was approved by the local authorities (Landesdirektion Dresden, 24–9165.40–4, 24.9168.21–4/2004–1). Luciferase-expressing PC3-PSCA cells (1 x 106) were injected s.c. into the right flank of experimental mice either alone or in the presence of 10 µg αPSCA TM and 1 × 106 UniCAR BB/ζ- or UniCAR 28/ζ-armed Tconvs. To investigate responsiveness to Treg suppression, 1 × 106 UniCAR BB/ζ-endowed Tregs were additionally added in two groups of mice. As reference value bioluminescence signal was determined 2 h after cell injection. For that purpose, mice were narcotized as described elsewhere.19 Next, 200 µl D-luciferin potassium salt (15 mg/ml) (ThermoFisher Scientific) was inoculated i.p. and the bioluminescence signal was measured by using an In-Vivo-Xtreme imaging system 10–15 min later (exposure time of 60 s, Bruker, Germany). Tumor burden was assessed over 19 days. Resulting data were quantified as previously published23 by using Bruker MI SE software (Bruker, Germany) and correlated to that of day 0.
8
Luciferase Assay in K562 Cells
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Luciferase assays were performed 3 days post electroporations; 0.5 × 105 K562 cells were removed and transferred to a black 96-well plate (ThermoFisher #237105). D-Luciferin, Potassium Salt (ThermoFisher #L2916) was added to each well to a final concentration of 150 μg/mL. Cells were shaken for 5 min prior to reading at 450 nm wavelength on a synergy 2 spectrophotometer (BioTek).
9
Niclosamide-based Compound Screening
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Niclosamide and Tween80® were purchased from Sigma-Aldrich (St. Louis, MO). Cycloheximide and D-luciferin potassium salt were from Thermo Fisher Scientific Inc. (Waltham, MA). PEG-8 CapRrylic/Capric glycerides (Labrasol® ALF), Plurol isostearique, and Labrafac lipophile WL 1349 were from Gattesfosse (Paramus, NJ).
10
In Vivo Luminescence Imaging Protocol
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Luminescence imaging was performed using a dedicated small animal multimodal imaging system (Xtreme, Bruker, Germany) 10 min after intraperitoneal (i.p.) injection of 200 μl of D-luciferin potassium salt (15 mg/ml) (Thermofisher, Dreieich, Germany). Optical imaging was collected with a 1-minute exposure and pseudo color representations of light intensity were superimposed over the reference image. To quantify the detected light, a region of interest was manually selected over the signal intensity and the light emitted from each region was evaluated and quantified after background subtraction. In parallel, an X-ray photograph was taken from the animals at the same position.
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