Lactate dehydrogenase (LDH) release from HaCaT spheroids was monitored by collecting aliquots of the medium using a standard spectrophotometric method [14 (link)]. The method is based on a coupled enzymatic reaction in which LDH catalyses the conversion of lactate to pyruvate via NAD+ reduction to NADH. Diaphorase reduces tetrazolium salt—oxidizing NADH in the process—to a red formazan product that can be measured at 490 nm. The medium derived from HaCaT spheroids, treated for 24 h with different concentrations of NaDES extracts (range in content of malvidin: 0.05–1.1 μg mL−1), was collected and the increase in absorbance between the treatment after 24 h and the control was monitored at 37 °C using an Allsheng FlexA-200 Microplate Reader. Menadione (50 μM for 16 h of treatment) was used as a cytotoxic agent [14 (link)].
Flexa 200 microplate reader
Manufactured by Allsheng
Sourced in China
The FlexA-200 Microplate Reader is a versatile laboratory instrument designed for the detection and quantification of various analytes in microplates. It features multiple detection modes, including absorbance, fluorescence, and luminescence, allowing for a wide range of applications in biochemical, cell-based, and molecular biology assays.
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Lab products found in correlation
Wst 8, by Dojindo Laboratories (1 mentions)
Epiquik global histone h3 acetylation assay kit, by Epigentek (1 mentions)
Epiquik global histone h4 acetylation assay kit, by Epigentek (1 mentions)
Pierce coomassie plus bradford assay, by Thermo Fisher Scientific (1 mentions)
Fitamp blood and cultured cell dna extraction kit, by Epigentek (1 mentions)
Methylflash methylated dna quantification kit, by Epigentek (1 mentions)
Methylflash hydroxymethylated dna quantification kit, by Epigentek (1 mentions)
Dna rna shield, by Zymo Research (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
5 protocols using flexa 200 microplate reader
1
Lactate Dehydrogenase Release Assay
2
Evaluating Cell Viability in 3D HaCaT Spheroids
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The cell viability was assessed by WST8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] (Dojindo Molecular Technologies, Japan), which, in the presence of an electron mediator, is reduced by dehydrogenases in cells (as a vitality biomarker) to formazan dye, which is soluble in the tissue culture medium. The amount of the formazan dye generated by dehydrogenases in cells is directly proportional to the number of living cells. The decrease in absorbance between the treatment after 24 h and the control was monitored at 37 °C at 450 nm using an Allsheng FlexA-200 Microplate Reader [15 (link)]. For this set of experiments, BET-CA formulation was added to the medium of human 3D HaCaT spheroids for 24 h (range in content of malvidin: 0.05–1.1 μg mL−1). The free radical-generating chemical menadione (50 μM for 16 h of treatment) was used as cytotoxic agent [14 (link)]. 24 h treatment with malvidin in BET-CA (range of 0.07–0.6 μg mL−1) was used as a reference.
3
Quantifying Histone H3 and H4 Acetylation
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Histone H3 and H4 acetylation were quantified using the EpiQuik™ Global Histone H3 Acetylation Assay Kit (Epigentek, Farmingdale, NY, USA) and the EpiQuik™ Global Histone H4 Acetylation Assay Kit (Epigentek, Farmingdale, NY, USA) according to the manufacturer’s instructions. First, histones were isolated from freshly isolated PBMCs using the reagents of the respective kit as well as trichloroacetic acid (TCA) and acetone. Histones were then quantified using the Pierce™ Coomassie Plus Bradford Assay (Thermo Scientific™, Waltham, MA, USA). Histone extracts were stored at −80 °C until assayed. Subsequently, 1 ug of protein was immobilized on a 96-well plate and acetylated H3/H4 was detected with capture and detection antibodies. The plate was read at 450 nm using the FlexA-200 microplate reader (Allsheng, Hangzhou, China), and OD was displayed directly.
4
DNA Methylation and Hydroxymethylation Profiling
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Freshly isolated PBMCs were reconstituted in a 10% (v/v) solution of DNA/RNA shield™ (Zymo Research™, Irvin, CA, USA) for further extraction of DNA and stored at −80 °C. DNA was extracted using the FitAmp™ Blood and Cultured Cell DNA Extraction Kit (Epigentek, Farmingdale, NY, USA). DNA extracts were used for quantification of methylation and hydromethylation using the Methylflash™ Methylated DNA Quantification Kit (Epigentek, Farmingdale, NY, USA) and the Methylflash™ Hydroxymethylated DNA Quantification Kit (Epigentek, Farmingdale, NY, USA). Briefly, 100 ng of sample DNA was immobilized on a 96-well plate with high DNA affinity. Methylated DNA and hydroxymethylated DNA were then detected using capture and detection antibodies against 5-mC and 5-hmC. The plate was read at 450 nm using the FlexA-200 microplate reader (Allsheng, Hangzhou, China). The absolute percentage of methylated or hydroxymethylated DNA was calculated from the optical density (O.D.) using the slope of a standard curve (50% methylated DNA or 25% hydroxymethylated DNA).
5
Extracellular Laccase Activity Assay
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The activity assay of extracellular laccase was determined at 25 °C by using 1.0 mM ABTS as substrate. The reaction mixture in a total volume of 3.0 mL contained 0.1 mL of laccase sample, 0.9 mL of 1.0 mM ABTS, and 2.0 mL of 100/200 mM citrate/phosphate at pH 4.5 (buffer A) solution. The increase of the OD at 420 nm in 3 min was measured by UV-9000 S spectrophotometer (Shanghai Metash Instruments Co., Ltd, China). One unit (U) of enzyme activity was defined as the amount of laccase that transformed 1 µmol ABTS per minute, and the laccase activity was expressed in the form of U/L. The soluble protein content of the enzyme sample was quantified according to Bio-Rad Protein Assay (Zheng et al. 2020 (link)) using bovine serum albumin (BSA) as a standard. A volume of 20 µL of the enzyme sample was incubated with 200 µL of Coomassie Brilliant Blue G-250 staining solution in a 96-well plate at 25 °C for 3–5 min. The absorbance was measured at 595 nm with FlexA-200 Microplate Reader (Hangzhou Allsheng Instruments Co, Ltd, China).
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