The MTS cell viability assay was performed as described previously (Hamada et al. 2021 (link); Arai et al. 2015 (link)). Briefly, 48 h after transfection with negative control siRNA and SPHK1- or LTB-specific siRNAs, cells were treated with CellTiter 96 Aqueous One Solution Reagent (Promega, Madison, WI). After 1 h of treatment, the optical density was measured at 490 nm on a GloMax®-Multi + Detection System Glomax (Promega). Results were presented as the mean ± standard deviation for three separate determinations.
Glomax multi detection system glomax
Manufactured by Promega
Sourced in United States
The GloMax®-Multi + Detection System is a versatile laboratory instrument designed for a wide range of luminescence-based assays. It provides accurate and reliable measurements of luminescent signals, serving as a core tool for various applications in life science research and development.
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6 protocols using glomax multi detection system glomax
1
MTS Cell Viability Assay for siRNA Transfection
2
MTS Assay for Cell Viability
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The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell viability assay was performed as described previously (29 (link)). Briefly, 48 h after transfection with negative control or ZNF132-specific siRNAs, cells were treated with CellTiter 96 Aqueous One Solution Reagent (Promega, Madison, WI). After 1 h of treatment, the optical density was measured at 490 nm on a GloMax®-Multi+ Detection System Glomax (Promega). Results were presented as the mean ± standard deviation for three separate determinations.
3
Caspase-Mediated Apoptosis Assay
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The apoptosis assay was performed as described previously (29 (link)). Briefly, 48 h after transfection with negative control or ZNF132-specific siRNAs, cells were treated with a Caspase-Glo 3/7 assay kit (Promega). After 1 h of incubation, the luminescent signal was measured on a GloMax-Multi+ Detection System Glomax (Promega). Results were presented as the mean ± standard deviation for three separate determinations.
4
Caspase-3/7 Apoptosis Assay
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The apoptosis assay was performed as described previously (Hamada et al. 2021 (link); Arai et al. 2015 (link)). Briefly, 48 h after transfection with negative control siRNA and SPHK1- or LTB-specific siRNAs, cells were treated with a Caspase-Glo 3/7 assay kit (Promega). After 1 h of incubation, the luminescent signal was measured on a GloMax-Multi + Detection System Glomax (Promega). Results were presented as the mean ± standard deviation for three separate determinations.
5
Assessing VHL as miR-429 Target
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To assess if VHL is a direct target of miR-429, pGL3-control vector or pGL3-VHL-WT was transfected with lipofectamine 3000 (Invitrogen) in SKBR3 cells, according to the manufacturer's suggestion, in combination with 100 nM mimic miR-429 or miR-142 (a known regulator of VHL) or scramble miR sequence. After 24 h, Gaussia Luciferase (GLuc) activity was measured in the collected medium and normalized on the Secreted Alkaline Phosphatase (SEAP) content. Results were detected by Glomax (GloMax-Multi Detection System; Promega, Madison, WI, USA).
6
Gauging miR675-5p Regulatory Role
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For the study of miR675-5p target, pGL3-control or pGL3-VHL-WT was transfected in U251 cells according to the manufacturer's instructions. After 24h, the medium was changed and the cells were treated for 18h with miR675-5p or miR21-5p and their relative scrambles. At the end of the treatment, the medium was collected and measured for Gaussia Luciferase (GLuc) and the transfection was normalized for the Secreted Alkaline Phosphatase (SEAP). Results were detected by Glomax (GloMax-Multi Detection System; Promega, Madison, WI, USA).).
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