The antiviral activity of investigated compounds was evaluated by measuring the IC50 values against the HIV-1 wild-type reference strain NL4-3 in a TZM-bl cell line-based phenotypic assay named BiCycle Assay [14 (link)]. The method includes a first round of infection in H9 cells at 0.08 MOI in the presence of the serial dilution of compounds in a 96-well plate. In each plate the reference compound, the mock control (uninfected cells) and the virus control were included. After 72 h, 50 µl of supernatants from each well were used to infect the TZM-bl cell line, which allows the quantitative analysis of HIV-1 infection by measuring the expression of the luciferase gene integrated in the genome of the cells under the control of the HIV-1 LTR promoter. After 48 h, dose–response curves were generated by measuring reporter gene expression in each well by using Bright-Glo Luciferase Assay (Promega) through the GloMax® Discover Multimode Microplate Reader (Promega). Relative luminescence units measured in each well were elaborated with the GraphPad PRISM software version 9 to calculate IC50 values.
Glomax discover multimode microplate reader
Manufactured by Promega
Sourced in United States
The GloMax® Discover Multimode Microplate Reader is a versatile lab equipment designed for detecting and quantifying various biological and biochemical assays. It can measure multiple detection modes, including luminescence, fluorescence, and absorbance, within a single microplate.
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Lab products found in correlation
Renilla glo r luciferase assay system, by Promega (12 mentions)
Celltiter glo 2.0 luminescent cell viability assay, by Promega (5 mentions)
Dual luciferase reporter assay system, by Promega (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Celltiter glo luminescent cell viability assay, by Promega (3 mentions)
Prism software version 9, by GraphPad (3 mentions)
Neurobasal a medium, by Thermo Fisher Scientific (3 mentions)
Janus platform, by PerkinElmer (2 mentions)
Calcein am, by Thermo Fisher Scientific (2 mentions)
4 mu glcnac 2, by Merck Group (2 mentions)
U bottom plate, by Corning (2 mentions)
Nanobret nano glo substrate, by Promega (2 mentions)
Psicheck 2, by Promega (2 mentions)
Double luciferase report system detection kit, by Promega (2 mentions)
Celltrace violet, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Bright glo luciferase assay, by Promega (1 mentions)
Pmirglo vector, by Promega (1 mentions)
60 protocols using glomax discover multimode microplate reader
1
HIV-1 Antiviral Activity Evaluation
2
Quantifying Galactocerebrosidase Activity in Nanoparticles
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To determine GALC activity in NP samples, 2 μl of NPs was added to 50 μl of assay buffer [50 mM sodium citrate, 125 mM NaCl, and 0.5% Triton X-100 (pH 4.5)]. Then, 50 μl of 1 mM 4-methylumbelliferyl-β-d -galactopyranoside in assay buffer was added. Samples were incubated at 37°C for 20 min, and 200 μl of stop solution (0.5 M glycine, 0.3 M NaOH) was added. Samples were plated into a 96-well multiplate for fluorescence, and fluorescence was measured with the Promega GloMax discover Multimode microplate reader with an excitation filter of 365 nm and an emission filter of 415 to 485 nm.
Activity yield (AY%) of NPs was calculated as the ratio between the specific activity of the sample (SANPs) and the specific activity of the unaltered GALC (SAGALC) as follows where the specific activities are defined as unit of enzyme (U) per microgram of enzyme (μgGALC)
One unit of enzyme (U) is defined as 1 nmol of substrate (nmolSUB) hydrolyzed in 1 hour
Activity yield (AY%) of NPs was calculated as the ratio between the specific activity of the sample (SANPs) and the specific activity of the unaltered GALC (SAGALC) as follows where the specific activities are defined as unit of enzyme (U) per microgram of enzyme (μgGALC)
One unit of enzyme (U) is defined as 1 nmol of substrate (nmolSUB) hydrolyzed in 1 hour
3
Conditional Parasite Growth Inhibition Assay
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Serial dilutions of aTc, atovaquone, quinine, and bafilomycin A were generated to yield final concentrations ranging from 40–0.462 nM, 25–0.048 nM, 432–0.84 nM, and 40–0.08 nM, respectively. Synchronous ring-stage PfRAP01 and PfRAP21 conditional knockdown lines as well as a control cell line expressing an aptamer-regulatable fluorescent protein were maintained in high aTc (500 nM), low aTc in the case of PfRAP01 (8 nM) and PfRAP21 (5 nM) or no aTc, and were distributed into 384-well assay plates (Corning®, 89176-442). Compounds were transferred to the parasite-containing plates using the Janus® platform (PerkinElmer). DMSO- and dihydroartemisinin-treatment (500 nM) served as reference controls. Growth inhibition was analyzed after 72 and 120 h using the Renilla-Glo(R) Luciferase Assay System (Promega, E2750) and the GloMax® Discover Multimode Microplate Reader (Promega). IC50 values were obtained from corrected dose-response curves and plotted using GraphPad Prism 8.
4
Luminescence-based Parasite Growth Assay
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Assessment of parasite proliferation rate in the presence and absence of aTc was carried out using luminescence as a readout of growth. In 96-well U-bottom BD Falcon™ plates, synchronous ring-stage parasites were set up in triplicate and cultured in the presence (0.5 μM) and absence of aTc and luminescence measured at 0 and 72 h by quantitating luminescence using the Renilla-Glo(R) Luciferase Assay System (Promega E2750) and the GloMax® Discover Multimode Microplate Reader (Promega). Parasite growth was determined from normalized RLuc values, with dihydroartemisinin- (DHA) treated (0.5 μM) samples as a no growth control. Data were analyzed using GraphPad Prism Version 8.
5
Cytolytic Potency Assay for CAR T Cells
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Tumor cytolysis was assessed by labeling of tumor cells with 2.5 µM CellTrace™ Violet (#C34571, Thermo Fisher) and analysis of viable cells after coincubation with CAR T cells by flow cytometry or with the calcein-AM release assay. In detail, target cells were incubated with calcein-AM at 10 μM (#65-0853-39, Thermo Fisher) for 30 min at 37 °C, washed thoroughly, then coincubated in triplicates in flat bottom 96-well microtiter plates (#CLS3997-50EA Corning, Germany) with CAR T cells at effector-to-target (E:T) cell ratios from 40:1 to 10:1. Additional triplicate wells were set up to assess spontaneous (target cells alone in complete medium) and maximum release (target cells in medium plus 9% Triton X-100). After 4 h at 37 °C in 5% CO2, samples were transferred to black-walled 96-well microtiter plates (#165305 Thermo Fisher) and analyzed using the GloMax® Discover multi-mode microplate reader (Promega) at excitation 475 nm and emission 500–550 nm. Data were expressed as arbitrary fluorescent units (AFU). Specific lysis was calculated by using the formula [(test release − spontaneous release)/(maximum release − spontaneous release)] × 100.
6
Luciferase Assay for miR-204 Binding Site
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The fragments of wild‐type PCAT6 and mutant PCAT6 (containing a 5‐bp mutation in the predicted binding site of miR‐204) were cloned into the pGEM‐T easy vector system (wt‐PCAT6, mut‐PCAT6; Cat. A1260, Promega, Madison, WI, USA). The primers were shown in Table S1 . HEK293 cells were seeded into a 24‐well plate and cultured overnight. The 0.5 μg of pGL4 luciferase expression vector (Cat. E6651, Promega), 0.5 μg wt‐PCAT6 or mut‐PCAT6 plasmid and 50 nM miR‐204 mimics or miR‐204 inhibitor (Cat.B03001, B02003, GenePharma) were cotransfected into HEK293 cells using 3 μL lipofectamine2000 (Invitrogen). After 48 hours transfection, Dual‐Luciferase Reporter Assay System (Cat. E1910, Promega) was used to perform the luciferase assays. The ratio of Firefly/Renilla luciferase activity was determined by GloMax® Discover Multimode Microplate Reader (Promega).
7
Evaluating LINC00992 and GOLM1 miRNA interactions
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Fragments of full-length LINC00992 with wild-type or mutant binding sites for miR-3935 and sequences of GOLM1 3’UTR containing wild-type or mutated miR-3935 binding sites were inserted into the pmirGLO vectors (Promega, Madison, WI, USA) for the construction of reporters (LINC00992-WT, LINC00992-MUT, GOLM1-WT, GOLM1-MUT). Then, the four reporters and miR-3935 mimics or miR-3935 inhibitor (GenePharma) were co-transfected into DU145 and PC3 cells applying lipofectamine2000 (Invitrogen), as needed. Forty-eight hours later, Dual-Luciferase Reporter Assay System (Promega) was employed for the examination of the luciferase activity. GloMax® Discover Multimode Microplate Reader (Promega) assessed the ratio of Firefly/Renilla luciferase activity and the activity of Renilla was the normalized control.
8
Caspase-3 Activity Assay Protocol
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Cells were washed twice with ice-cold PBS. Seventy microliters ice-cold lysis buffer (25 mM HEPES, 5 mM MgCl2, 1 mM EGTA, 0.1% [v/v] Triton X-100) were added, and the samples were stored at −80°C. After thawing on ice, the lysates were centrifuged (14,000 × g, 10 min, 4°C) and 10 μL of the supernatant were transferred to a black 96 well plate (TPP). 90 μL substrate solution (55 μM of fluorogenic substrate Ac-DEVD-AFC (Enzo, #ALX-260-032-M005) 50 mM HEPES, 0.1% [w/v] CHAPS, 1% [w/v] sucrose, 10 mM DTT, pH 7.5) were added, and the production of free 7-amino-4-trifluoro-methyl coumarin (AFC) at 37°C was determined by fluorescence measurement (excitation: 405 nm; emission: 495-505 nm) using a GloMax® Discover Multimode Microplate Reader (Promega).
9
Measurement of Chitinase Activity
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We determined the chitinolytic activity using a synthetic fluorogenic substrate, 4-methyl umbelliferyl β-D-N, N′-diacetyl chitobioside [4-MU-(GlcNAc)2] (Sigma-Aldrich), as described previously.35 We measured the fluorescence of liberated 4-methyl umbelliferon using a GloMax Discover Multimode Microplate Reader (Promega, Madison, WI, USA) with excitation at 365 nm and emission at 445 nm.
To determine the optimal pH for chitinase activity, the enzyme was incubated with the 4-MU-(GlcNAc)2 substrate in 0.1 M Gly-HCl buffer (pH 1.0–3.0) or McIlvaine’s buffer (0.1 M citric acid and 0.2 M Na2HPO4; pH 2.0–8.0) at 37°C for 30 min.
To determine the optimal pH for chitinase activity, the enzyme was incubated with the 4-MU-(GlcNAc)2 substrate in 0.1 M Gly-HCl buffer (pH 1.0–3.0) or McIlvaine’s buffer (0.1 M citric acid and 0.2 M Na2HPO4; pH 2.0–8.0) at 37°C for 30 min.
10
Quantification of ROS Production
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ROS production was quantified and evaluated qualitatively by means of the fluorescent oxidant-sensing probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA, Invitrogen). After a 24-hour incubation period with the NCs and stimulation with LPS, the medium was removed, and a solution containing the fluorescent probe in PBS (5 µmol/L) was added (200 µL). After 5 minutes, inflammatory mediator expression was measured using a multimode plate reader (GloMax® Discover Multimode Microplate Reader, Promega, São Paulo, SP, Brazil). In the qualitative analyses, the samples were plated on 24-well plates (105 cells/well) in the bottom of the compartments, and the same protocol was followed. After 24 hours, the culture medium was removed, and the samples were analyzed and photographed by fluorescence microscopy (Invitrogen Evos FL Imaging System, Invitrogen). The value readings were normalized to LPS control cultures (LPS-100%), and the differences between mean values were statistically analyzed. The positive control group contained LPS-stimulated and untreated cells, and the negative control group contained cells maintained in DMEM (no LPS stimulation).
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