The TZM-bl assay (38 (link)) was performed in 96-well black or white plates using a firefly luciferase reporter assay kit (PromoCell GmbH, Germany). Briefly, 50 µL of cells (105/mL) were seeded in the 96 wells and incubated for 24 h at 5% CO2 and 37 °C. Diluted drugs mixed with viruses (25 ng/mL) plus diethyl aminoethyl-dextran (25 μg/mL) at final concentration were added to the cells (total final volume: 200 µL). After 48 h incubation, the supernatant medium switched to lysis buffer was shaken for 15 min, and d -luciferin was added to each well. Luciferase intensity was measured using a FLUOstar Omega instrument (BMG Labtech GmbH, Germany).
Fluostar omega instrument
Manufactured by BMG Labtech
Sourced in Germany
The FLUOstar Omega is a multi-mode microplate reader designed for a wide range of applications in life science research. It features flexible detection technologies, including fluorescence intensity, absorbance, and luminescence measurements. The instrument provides accurate and reliable data across various sample types and microplate formats.
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Lab products found in correlation
3 3 5 5 tetramethylbenzidine (tmb), by Thermo Fisher Scientific (3 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Camp glotm assay, by Promega (1 mentions)
Water soluble coelenterazine, by Nanolight Technology (1 mentions)
Cytation 3, by Agilent Technologies (1 mentions)
Spectramax m3, by Molecular Devices (1 mentions)
Sterile pestle, by Corning (1 mentions)
Fluorescence based enzymatic detection kit, by Abcam (1 mentions)
Accuri c6, by BD (1 mentions)
Psicheck 2, by Promega (1 mentions)
4d nucleofector kits, by Lonza (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Gblock, by Integrated DNA Technologies (1 mentions)
9 protocols using fluostar omega instrument
1
TZM-bl Luciferase Assay for Antiviral Screening
2
Quantifying SARS-CoV-2 RBD Binding
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Neu5Acα2-3Galβ1-3GlcNAc-PAA (Lectinity Holdings, Russia) was coated in a 96-well Nunc MaxiSorp plate (Sigma-Aldrich, Germany) at 0.5 μg/well overnight at 4°C, followed by blocking with 3% bovine serum albumin (BSA) (Sigma, Germany) in phosphate-buffered saline (PBS)-0.1% Tween 20 overnight. RBD proteins were preincubated with Strep-Tactin HRP antibody (IBA, Germany) (1:200) for 30 min on ice. Indicated protein amounts were diluted in PBS and applied onto the coated well, followed by incubation for 2 h at room temperature. TMB (3,3′,5,5′-tetramethylbenzidine; Thermo Scientific, Netherlands) substrate was used to visualize binding, after which the reaction was terminated using 1 M H2SO4. The optical density at 450 nm was measured in a FLUOstar Omega instrument (BMG Labtech), and MARS data analysis software was used for data analysis. Statistical analysis was performed using a two-way analysis of variance (ANOVA).
3
SARS-CoV-2 S1 Protein Binding Assay
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Galβ1,4GlcNAcβ1,3Galβ1,4GlcNAc (Consortium for Functional Glycomics) was coated in a 96-well Nunc MaxiSorp plate (Sigma-Aldrich) at 0.5 µg/well overnight at 4°C, followed by blocking with 3% bovine serum albumin (BSA; Sigma) in PBS–0.1% Tween. S1 proteins were preincubated with Strep-Tactin–horseradish peroxidase (HRPO) (1:200) for 30 min on ice. For each protein, 2-fold dilutions were made in triplicate in PBS and applied onto the coated well, followed by incubation for 2 h at room temperature. TMB (3,3′,5,5′-tetramethylbenzidine; Thermo Scientific) substrate was used to visualize binding, after which the reaction was terminated using 1 M H2SO4. The optical density at 450 nm (OD450) was measured in a FLUOstar Omega instrument (BMG Labtech), and MARS data analysis software was used for data analysis. Statistical analysis was performed using a two-way analysis of variance (ANOVA).
4
Verifying MC3R Overexpression via cAMP
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The presence and functionality of MC3R after
induced overexpression were verified using the cAMP-GloTM Assay (Promega
Corporation, Madison, WI). After the cells were prepared as described
above, the doxycyclin-containing medium was replaced with a treatment
solution containing 500 μM IBMX and 100 μM Ro 20-1724
as well as 0, 0.5, 5, 50, or 500 nM of γ-MSH, or 4 μM
cAMP in PBS as a positive control. The plate was then incubated at
37 °C for 1 h, after which the assay was performed according
to the manufacturer’s protocol. Briefly, cell lysis was followed
by incubation with a cAMP detection solution containing Protein Kinase
A at room temperature for 20 min and then with Kinase-Glo Reagent
at room temperature for 10 min. Finally, the luminescence was measured
using a FLUOstar Omega instrument (BMG Labtech, Ortenberg, Germany).
induced overexpression were verified using the cAMP-GloTM Assay (Promega
Corporation, Madison, WI). After the cells were prepared as described
above, the doxycyclin-containing medium was replaced with a treatment
solution containing 500 μM IBMX and 100 μM Ro 20-1724
as well as 0, 0.5, 5, 50, or 500 nM of γ-MSH, or 4 μM
cAMP in PBS as a positive control. The plate was then incubated at
37 °C for 1 h, after which the assay was performed according
to the manufacturer’s protocol. Briefly, cell lysis was followed
by incubation with a cAMP detection solution containing Protein Kinase
A at room temperature for 20 min and then with Kinase-Glo Reagent
at room temperature for 10 min. Finally, the luminescence was measured
using a FLUOstar Omega instrument (BMG Labtech, Ortenberg, Germany).
5
Bioluminescent Enzyme Assay for Dox Induced Gene Expression
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Isolate DSY5624 was cultured overnight in YEPD and grown to log phase for 4 hr in the same medium (5 ml) containing different Dox concentrations. After incubation, cells were washed and resuspended in 200 μl R-luc buffer (0.5 M NaCl, 0.1 M Na2HPO4 pH 7.0, 1 mM EDTA). Eighty μl of each suspension was pipetted in duplicates into black half area 96-well plate. Water-soluble coelenterazine (catalog # 3032, Nanolight Technology, Pinetop, AZ, USA) was resuspended in 5 mL PBS (phosphate buffered saline: 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.47 mM KH2PO4) and kept in the dark. Plates were placed in a FLUOstar Omega instrument (BMG Labtech, Champigny/Marne, France). The instrument was kept at a temperature of 30°C. After shaking the plate for 3 s and injection of 20 μl coelenterazine, reading of luminescence was started and continued with intervals of 1.5 s for 50 s.
6
High-Throughput Mycobacterial Luminescence Assay
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All experiments
were performed with biological triplicates, unless otherwise indicated.
For experiments with live Mtb, measurements were
made of independent bacterial cultures. For experiments with recombinant
Hip1, measurements were made of independent mixtures using the same
enzyme preparation. All chemiluminescence assays were performed in
white, opaque flat-bottom 384- or 96-well plates. Luminescence was
measured in different microplate readers, depending on the laboratory
location. Measurements of H37Rv, mc26020, and M.
marinum were obtained on a SpectraMax M3 instrument (Molecular
Devices) at 25 °C. Measurements of recombinant Hip1, mc26020, NTMs, and other bacteria were obtained on a Cytation 3 instrument
(Biotek) at 37 °C. Measurements of rifampicin susceptibility
of the H37Rv and RpoB H526D mutant were obtained on a FLUOstar Omega
instrument (BMG Labtech) at 37 °C. For all experiments, luminescence
measurements began within 5 min after the addition of the FLASH probe
and were continued for at least 1 h. Measurements were made without
an emission filter, using a 1 s integration time. For each sample,
luminescence measurements from the first hour were summed to yield
integrated luminescence.
were performed with biological triplicates, unless otherwise indicated.
For experiments with live Mtb, measurements were
made of independent bacterial cultures. For experiments with recombinant
Hip1, measurements were made of independent mixtures using the same
enzyme preparation. All chemiluminescence assays were performed in
white, opaque flat-bottom 384- or 96-well plates. Luminescence was
measured in different microplate readers, depending on the laboratory
location. Measurements of H37Rv, mc26020, and M.
marinum were obtained on a SpectraMax M3 instrument (Molecular
Devices) at 25 °C. Measurements of recombinant Hip1, mc26020, NTMs, and other bacteria were obtained on a Cytation 3 instrument
(Biotek) at 37 °C. Measurements of rifampicin susceptibility
of the H37Rv and RpoB H526D mutant were obtained on a FLUOstar Omega
instrument (BMG Labtech) at 37 °C. For all experiments, luminescence
measurements began within 5 min after the addition of the FLASH probe
and were continued for at least 1 h. Measurements were made without
an emission filter, using a 1 s integration time. For each sample,
luminescence measurements from the first hour were summed to yield
integrated luminescence.
7
Metabolite Measurement in Drosophila Larvae
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Measurements of total body glucose and lactate were performed on a pool of 5 larvae per genetic condition using a fluorescence-based enzymatic detection kit (Biovision Inc.) as described before [81 (link)]. Briefly, larvae were collected in 1.5 ml microcentrifuge tubes, the excess of water was removed and samples were frozen stored at -80°C until further analysis. Upon defrosting, 15 μl PBS per larvae was added and tissues were disrupted with a sterile pestle (Axygen), on ice. Samples were spun at 14,000 x g for 10 min, at 4°C. The supernatant was immediately used for metabolite measurements according to manufacturers’ protocol. Fluorescence readings (Ex/Em 535/590) were taken with the FLUOstar Omega instrument (BMG Labtech). Results were read from a linear regression curve based on the dilutions of the standard. Statistical analysis between indicated groups was performed using the unpaired t-test and considered significant at P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***).
8
Folate-based Fluorescence Imaging of T-ALL
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The fluorescent folate analogues folate-FITC (FL-FITC) and folate-TAMRA (FL-TAMRA) were synthesized as described in the Supplemental Material. T-ALL cells were grown in the presence or absence of FL-FITC, and after treatment, cells were washed in cold PBS containing 2 mmol/L EDTA. Background fluorescence on the cell surface due to nonspecific FA fluorescence was determined in each experiment by incubating cells with free FA. For internalization studies, T-ALL cells were incubated for 6 h at 37°C and subsequently either washed as described above or subjected to a short acidic cold wash with PBS, 50 mM glycine pH 4.0 to eliminate cell surface-bound fluorescence. Fluorescent signal was assessed using flow cytometry with an Accuri C6 (BD). A minimum of 10,000 events was collected for each biological sample. TAMRA fluorescence was assessed using a Fluostar Omega instrument (BMG-labtech) with excitation set at 541 wavelength and emission at 568 nM.
9
HERV DNA-Driven Luciferase Reporting
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To construct luciferase reporter plasmids, we cloned synthetic fragments of HERV DNA (sequences in Table S8) using gBlocks ® (Integrated DNA Technologies) into psiCHECK-2 (Promega). The fragments were inserted downstream of the Renilla luciferase gene using XhoI and NotI restriction sites and DNA ligation. Plasmid inserts were verified by sequencing (Macrogen).
Luciferase Reporter Assay 5x10 5 HAP1 cells were transiently transfected with 300 ng of plasmid using the SE cell line 4D-Nucleofector kit S (Lonza), on a 4D nucleofection unit (Lonza) using protocol DZ-113. Cells were plated out evenly in three separate wells on a 12-well plate, cultured for 24 hours, and luciferase reporter activity was assessed using Dual Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol. Luminescence measurements were made in a FLUOstar Omega instrument (BMG LABTECH).
Luciferase Reporter Assay 5x10 5 HAP1 cells were transiently transfected with 300 ng of plasmid using the SE cell line 4D-Nucleofector kit S (Lonza), on a 4D nucleofection unit (Lonza) using protocol DZ-113. Cells were plated out evenly in three separate wells on a 12-well plate, cultured for 24 hours, and luciferase reporter activity was assessed using Dual Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol. Luminescence measurements were made in a FLUOstar Omega instrument (BMG LABTECH).
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