Uncoated 2HB 96-well plates (Thermo Fisher Scientific) were coated with 0.5 μg/mL SIVmac239 gp120 (Immune Technology, New York, NY) in coating buffer (2.25mg/mL Na2CO3 and 4.395mg/mL NaHCO3 in distilled H2O). Plates were incubated overnight at 4°C. The next day, plates were washed with wash buffer (1X PBS with 0.05% V/V TWEEN-20 [Thermo Fisher Scientific]) and blocked in a buffer containing 1X PBS supplemented with 10% FBS for one hour at room temperature. After blocking, plates were washed and serial dilutions of plasma were added for 90 minutes at room temperature. Plasma samples were heat-inactivated at 56°C for 30 minutes, prior to use. Serial dilutions of anti-SIV gp120 monoclonal antibody (B404, NIH HIV Reagent Program, Division of AIDS, NIAID, NIH) were plated as a positive control. Next, plates were washed with wash buffer and anti-monkey IgG HRP (SouthernBiotech, Birmingham AL) was diluted 1:10,000 in blocking buffer and added to each well. Plates were incubated for one hour at room temperature. Plates were then washed with wash buffer and TMB substrate (3,3’5,5’ – tetramethylbenzidine, Thermo Fisher Scientific) was added to each well. After a 15 minute incubation, HCl (1N) was added to each well to stop the reaction. ELISA plates were immediately read using a GloMax®-Multi Detection System microplate reader (Promega, Madison WI) at 450 nm absorbance.
Glomax multi detection system microplate reader
Manufactured by Promega
Sourced in United States, Italy
The Glomax Multi Detection System is a microplate reader designed to measure various luminescent, fluorescent, and absorbance-based assays. It offers a versatile platform for a wide range of applications in life science research and provides accurate and reliable data.
Automatically generated - may contain errors
Lab products found in correlation
Anti monkey igg hrp, by Southern Biotech (2 mentions)
Uncoated 2hb 96 well plates, by Thermo Fisher Scientific (2 mentions)
V 5 tween 20, by Thermo Fisher Scientific (2 mentions)
Tetramethylbenzidine, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Lactate glo assay kit, by Promega (1 mentions)
8 protocols using glomax multi detection system microplate reader
1
ELISA Assay for SIV gp120 Antibodies
2
Cell Proliferation and Viability Assay
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MTT assays were performed to monitor cell proliferation and
viability (10 (link),11 (link)). Cells were seeded at 5000 cells/cm2 in 96-well
plates in standard culture medium. The final concentration
was 0.5 mg/mL MTT in standard culture medium after 24
h, 48 h, and 72 h of culturing. Following 4 h of incubation,
the MTT solution was removed and dimethyl sulfoxide was
added to dissolve the formed formazan crystals. The culture
dishes were agitated in a shaker for 10 minutes to ensure the
dissolution of formazan crystals. Absorbance was measured at
a wavelength of 560-750 nm using a Glomax Multi Detection
System microplate reader (Promega, USA) (twelve replicates
for each treatment).
viability (10 (link),11 (link)). Cells were seeded at 5000 cells/cm2 in 96-well
plates in standard culture medium. The final concentration
was 0.5 mg/mL MTT in standard culture medium after 24
h, 48 h, and 72 h of culturing. Following 4 h of incubation,
the MTT solution was removed and dimethyl sulfoxide was
added to dissolve the formed formazan crystals. The culture
dishes were agitated in a shaker for 10 minutes to ensure the
dissolution of formazan crystals. Absorbance was measured at
a wavelength of 560-750 nm using a Glomax Multi Detection
System microplate reader (Promega, USA) (twelve replicates
for each treatment).
3
Evaluation of Melanoma Cell Viability
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Using published protocols [38 (link), 80 (link), 82 (link)–84 (link)], cell viability of mouse and human melanoma cells was evaluated by MTT analysis. Each experimental condition was replicated in 8 wells. Cells were then washed and fresh medium containing MTT (0.5 mg/ml) was added in each well. After 4 h incubation at 37°C, the supernatant was gently removed and formazan crystals were dissolved in DMSO. Absorbance was recorded at 570 nm with correction at 690 nm using a Glomax Multi Detection System microplate reader (Promega, Milano, Italy).
4
Measuring Cell Viability via MTT Assay
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Cell viability was determined by MTT assay using published protocols [29 (link),31 (link),51 (link),52 (link)]. Briefly, the assay was performed treating cells for 24 h in the absence (vehicle) or in the presence of increasing concentrations of compounds. MTT absorbance was quantified spectrophotometrically using a Glomax Multi Detection System microplate reader (Promega, Milan, Italy).
EC50 (the concentration producing half the maximum effect) and Emax concentration (producing the maximum effect) were determined by nonlinear regression curve analysis of the concentration–effect responses. Differences in potency values among concentration–response curves were calculated with the F-test. The GraphPad Prism software package (GraphPad Software, San Diego, CA, USA) was used.
EC50 (the concentration producing half the maximum effect) and Emax concentration (producing the maximum effect) were determined by nonlinear regression curve analysis of the concentration–effect responses. Differences in potency values among concentration–response curves were calculated with the F-test. The GraphPad Prism software package (GraphPad Software, San Diego, CA, USA) was used.
5
Cell Viability Assay of Glioma Cells
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Cell viability of human and mouse glioma cells in the presence of CDDP at increasing concentrations for 24 h was evaluated by MTT analysis (40 (link), 45 (link), 46 (link)). MTT (Sigma-Aldrich, Saint Louis, MO, USA) absorbance was quantified spectrophotometrically using a Glomax Multi Detection System microplate reader (Promega, Madison, WI, USA).
6
SIVmac239 gp120 ELISA Assay
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Uncoated 2HB 96-well plates (Thermo Fisher Scientific) were coated with 0.5 μg/mL SIVmac239 gp120 (Immune Technology, New York, NY) in coating buffer (2.25mg/mL Na2CO3 and 4.395mg/mL NaHCO3 in distilled H2O). Plates were incubated overnight at 4°C. The next day, plates were washed with wash buffer (1X PBS with 0.05% V/V TWEEN-20 [Thermo Fisher Scientific]) and blocked in a buffer containing 1X PBS supplemented with 10% FBS for one hour at room temperature. After blocking, plates were washed and serial dilutions of plasma were added for 90 minutes at room temperature. Plasma samples were heat-inactivated at 56°C for 30 minutes, prior to use. Serial dilutions of anti-SIV gp120 monoclonal antibody (B404, NIH HIV Reagent Program, Division of AIDS, NIAID, NIH) were plated as a positive control. Next, plates were washed with wash buffer and anti-monkey IgG HRP (SouthernBiotech, Birmingham AL) was diluted 1:10,000 in blocking buffer and added to each well. Plates were incubated for one hour at room temperature. Plates were then washed with wash buffer and TMB substrate (3,3’5,5’–tetramethylbenzidine, Thermo Fisher Scientific) was added to each well. After a 15 minute incubation, HCl (1N) was added to each well to stop the reaction. ELISA plates were immediately read using a GloMax-Multi Detection System microplate reader (Promega, Madison WI) at 450 nm absorbance.
7
Lactate Quantification in MoDCs
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The concentration of lactate in supernatant of M8-treated MoDCs was measured using a luminometric Lactate-Glo assay kit (Promega), according to the manufacturer’s protocol. The luminometry was measured with GloMax®-Multi Detection System microplate reader (Promega). Results were quantified using a standard curve.
8
DENV-2 NS2B/NS3 Protease Inhibition Assay
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The in vitro protease inhibition assay was carried out using purified DENV-2 NS2B/NS3 protease as the enzyme and Boc-Gly-Arg-Arg-MCA as the substrate according to the method previously described by our group (Salleh et al., 2019) . The concentration of the enzyme and the substrate were 0.5 μM and 10mM, respectively in 200 mM of Tris-HCl buffer (pH 8.5) while the concentration of the inhibitor was 200 μg/mL. All of the tests were performed in quadruplicates. Firstly, the Tris-HCl buffer (pH 8.5) was pipetted into the wells, followed by 1 μL of the inhibitor and 3.1 μL of the enzyme. Before adding the substrate, the enzyme and the inhibitor were incubated at 37°C for 10 minutes. After adding the substrate, the reaction mixture was incubated at 37°C for 60 minutes. All of the reactions were performed in 96-well plates with a final volume of 100 μL per well. Fluorescence was detected using a Promega Glomax Multi Detection System microplate reader with the excitation and emission wavelengths at 365 and 410-460 nm, respectively. In order to determine the IC 50 values, the same protocol was used as described above with serial dilutions of the inhibitors with concentrations in the range of 1.5625 to 200 μg/mL.
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