The reporter plasmids phRL-TK rs2734647C, or rs2734647T, respectively, were constructed by cloning a 3′UTR fragment of 346 bp and including the SNP downstream of the Renilla luciferase open reading frame, making use of the XbaI restriction site of phRL-TK (forward primer: 5′-ATTATCTAGACCAGGTCTACCCCTCCCGGC-3′, reverse primer: 5′-ATTATCTAGAGGCTGCTCCCTGTCCCAGGT-3′). Sequence integrity was verified using Sanger sequencing. Of the Renilla luciferase reporter construct (phRL-TK rs2734647C, or rs2734647T, respectively), 1 μg (per well), plus 1 μg (per well) of the reference construct pCMV-LacZ (Clontech, Mountain View, CA, USA) were co-transfected in the presence of 10 pg of mirVana miRNA mimic hsa-miR-4711-3p, hsa-miR-511, hsa-miR-515-3p, hsa-miR-519e-3p, negative control #2 (all Life Technologies, Darmstadt, Germany), or no miRNA, respectively. After 24 h, cells were split in 96-well plates, creating four technical replicates for each condition, and separately for luciferase and beta-galactosidase measurement. Enzyme activity was determined using a Mithras LB 940 Plate Reader (Berthold, Bad Wildbad, Germany). Renilla luciferase activity was normalized to beta-galactosidase activity.
Lb940 plate reader
Manufactured by Berthold Technologies
Sourced in Germany
The LB940 plate reader is a multi-mode microplate reader that can perform various detection methods such as absorbance, fluorescence, and luminescence. It is designed for a wide range of applications in life science research, including cell-based assays, enzyme activity measurements, and nucleic acid quantification.
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4 protocols using lb940 plate reader
1
Characterization of miRNA's Effect on Renilla Luciferase
2
Cell Viability Assay Protocol
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Cell growth was assayed using the Cell‐Titer Glo Luminescent Cell Viability Assay (Promega, Madison WI, USA, #G7573) following the manufacturer's protocol. Plates were read on a Berthold Mithras LB 940 Plate Reader.
3
Protein Quantification and SDS-PAGE
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The protein concentration of cell lysates was determined using a BCA Assay kit (Thermo Fisher Scientific, Dreieich, Germany) as described in the supplier's protocol, and measured at 560 nm wavelength (OD560) with LB940 plate reader (Berthold Technologies). Aliquots (20-45 μg) of protein samples were heat-treated at 95 o C for 5 min with loading buffer (125 mM Tris-HCl pH 6.8, 2% SDS, 50% Glycerin, 0.4% Bromophenol Blue, 5% β-Mercaptoethanol) and were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% SDS-gel for 90 minutes at a constant voltage of 120 V. Subsequently, the gels were used for Western Blotting.
4
Analyzing Fas-Triggered Protein Complexes
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For analysis of Fas-triggered protein complexes, cells were trypsinized, and suspended cells (2 × 106) were stimulated with anti-CD95 (CH-11; 1 μg/ml) for various time intervals at 37°C and lysed in lysis buffer (10 mM HEPES, pH 7.0, 150 mM NaCl, 1% Triton X-100, containing 0.1% Phosphatase Inhibitor-cocktail II and III, 0.1% Phosphatase Inhibitor-cocktail II, 0.25% Protease Inhibitor-cocktail EDTA-free) for 1 hour on ice. In control cells, CH-11 was added to lysates at a final concentration of 1 μg/ml to immunoprecipitate unstimulated Fas receptors. After centrifugation at 15,000g for 10 minutes, supernatants were precipitated with 20 μl goat antimouse IgM-Sepharose overnight at 4°C. Protein concentration in lysates from stimulated cells was determined using a BCA Assay kit (Thermo Fisher Scientific, Dreieich, Germany) as described in the supplier's protocol, and the Berthold LB940 plate reader. Cell lysates (500 μg) were incubated with anti-CD95 (CH-11), goat anti-ADAM15, anti-Src, and anti-CaM antibodies (1 μg/ml each) overnight at 4°C and precipitated using IgM-sepharose (Sigma) for CH-11 and protein G-agarose for all other antibodies, washed 3 times in lysis buffer, and analyzed by immunoblotting.
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