For cell growth assessment, HCC cells were seeded at 7500 cells per well in 96-well plates 24 h after transfection. Cell viability was measured at the indicated times using Cell Counting Kit-8 (Dojindo) according to the manufacturer’s instructions (WST-8 assay). The absorbance at 450 nm was measured using a Synergy H4 Microplate Reader system (BioTek). For the evaluation of sorafenib cytotoxicity, miR-493-5p mimic and control mimic-transfected cells were seeded in 96-well plates (10,000 cells/well). The next day, the medium was changed, and the cells were cultured in medium containing different concentrations of sorafenib for 48 h. Cell viability was measured as mentioned above. Sorafenib (Nexavar; PubChem CID: 216239) was purchased from Santa Cruz Biotechnology (#SC-220125).
Synergy h4 microplate reader system
Manufactured by Agilent Technologies
The Synergy H4 Microplate Reader system is a versatile and highly sensitive instrument designed for a wide range of applications in life science research and drug discovery. The core function of the Synergy H4 is to accurately measure and analyze various types of samples, such as cell-based assays, biochemical assays, and ELISAs, using multiple detection modes, including absorbance, fluorescence, and luminescence.
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6 protocols using synergy h4 microplate reader system
1
Cell Viability and Sorafenib Cytotoxicity Assay
2
Time-Dependent Cytotoxicity of 5-AZA in HCC Cells
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For the evaluation of the time- and dose-dependent cytotoxicity of 5-AZA, HCC cells were seeded at 10,000 cells/well in 96-well plates. The next day, the medium was changed and cells were treated with the indicated concentrations of 5-AZA for 1–5 days. Cell viability was measured at the indicated times using the Cell Counting Kit-8 (Dojindo), according to the manufacturer’s instructions (MTT assay). The absorbance at 450 nm was measured using the Synergy H4 Microplate Reader system (BioTek). For the evaluation of sorafenib cytotoxicity, reconditioned and control cells were seeded in 96-well plates (10,000 cells/well). The next day, the medium was changed, and the cells were cultured in medium containing different concentrations of sorafenib for 48 hr. Treatment with 5-AZA was discontinued 2 days before seeding in 96-well plates, and cells were maintained without 5-AZA until the end of the experiments. Cell viability was measured as mentioned above.
3
Dual Luciferase Assay for IGF2 3'-UTR
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Dual luciferase reporter plasmids were purchased from GeneCopoeia, where the IGF2 3′-UTR (cat. #CS-MIT234L-MT01) and mutated IGF2 3′-UTR sequences (cat. #CS-MI235L-MT01) were cloned into a pEZX-MT01 vector. Renilla luciferase activity driven by a CMV promoter was used for normalization. Simultaneous cell transfections with 3′-UTR constructs (3 µg) and miR-493-5p mimics (100 mM) were performed in 35-mm-diameter dishes following the experimental procedure described above. Mutated IGF2 vectors and control miRNA mimics were used as negative controls. Cells were collected 24 h after transfection, and protein was extracted using M-PER (Thermo Scientific). The firefly and Renilla luciferase activities were assayed with a Dual-Glo Luciferase Assay System (Promega) using a Synergy H4 Microplate Reader system (BioTek) as recommended by the manufacturer.
4
Evaluating Cytotoxicity of 5-AZA and GEM
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For the evaluation of the time and dose-dependent cytotoxicity of 5-AZA, PANC-1 cells were seeded at 7,500 cells per well in 96-well plates and Capan-2, PL45, and SU.86.86 cells were seeded at 10,000 cells/well (6 wells/condition). The next day, the medium was changed and cells were treated with the indicated concentrations of 5-AZA for one to five days (daily replacement). Cell viability was measured at the indicated times using the Cell Counting Kit-8 (Dojindo), according to the manufacturer’s instructions (MTT assay). The absorbance at 450 nm was measured using the Synergy H4 Microplate Reader system (BioTek). For the evaluation of concentration-dependent cytotoxicity of GEM (IC50), reprogrammed and control PANC-1 cells were seeded in 96-well plates (10,000 cells/well; 6 wells/condition). The next day, the medium was changed, and reprogrammed and control PANC-1 cells were cultured in medium containing different concentrations of GEM for 48 h. Treatment with 5-AZA was discontinued two days before seeding in 96-well plates, and cells were maintained without 5-AZA until the end of the experiments. Cell viability was measured as mentioned above. A similar protocol was used for the assessment of the effect of the SST analog on PANC-1 cell growth before and after epigenetic reprogramming.
5
Cell Growth Assessment via WST-8 Assay
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For the cell growth assessment, Hep3B cells were seeded at 10 000 cells per well in 96‐well plates 24 hours after transfection with MYCN siRNAs or miR‐493‐5p mimics and MYCN expression vector. Cell viability was measured at the indicated times by WST‐8 assay using a CCK‐8 (Dojindo). After cell culture medium was removed, 10 µL WST‐8 reagent and 100 µL fresh medium were added to each well. Cells were incubated at 37°C for 1 hour. The absorbance at 450 nm was measured using a Synergy H4 Microplate Reader system (BioTek).
6
Dual-Luciferase Assay for MYCN 3'UTR Regulation
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Dual luciferase reporter plasmids were purchased from GeneCopoeia, where miR‐493‐5p binding sites #1 and #2 from the MYCN 3′‐UTR were cloned downstream of the firefly luciferase reporter gene into pEZX‐MT01 and pEZX‐MT06 vectors, respectively. Renilla luciferase activity driven by a CMV promoter was used for normalization. Simultaneous cell transfections with 3′‐UTR constructs (3 µg) and miR‐493‐5p mimics (100 nmol/L) were undertaken following the experimental procedure described above. Mutated MYCN vectors (sites #1 and #2) and control miRNA mimics were used as negative controls. HepG2 cells were collected after transfection, and protein was extracted using M‐PER (Thermo Fisher Scientific). The firefly and Renilla luciferase activities were assayed with a Dual‐Glo Luciferase Assay System (Promega), using a Synergy H4 Microplate Reader system (BioTek). The ratio of firefly : Renilla luminescence was calculated for each well and expressed as dual luciferase activity.
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