Human MSCs cultured in ODM were transfected with miR-130a mimics or inhibitor, miR-27b mimics or inhibitor and its negative controls (Applied Biosystems/Ambion, Austin, TX, United States) using the Lipofectamine 3000 transfection agent (Invitrogen, Carlsbad, CA, United States). The alkaline phosphatase activity was measured using an assay kit (Colorimetric; Abcam). At indicated times, the conditioned medium was collected. Into a 96-well plate, 80 μl of samples was added followed by the addition of 50 μl of a 5 mM pNPP solution to each well. The reaction mixture was incubated at 25°C for 60 min and the reaction was stopped by adding 20 μl of the stop solution followed by gently shaking of the plate. The p-nitrophenol product, which was generated via enzymatic hydrolysis of the p-nitrophenylphosphate substrate, was detected at OD 405 nm using a microplate reader synergy HTX Multi-Mode Reader (BioTek Instruments, VT, United States).
Htx multi mode reader
Manufactured by Agilent Technologies
Sourced in United States
The HTX multi-mode reader is a versatile laboratory instrument designed for a wide range of applications. It is capable of performing absorbance, fluorescence, and luminescence measurements to support various experimental workflows.
Automatically generated - may contain errors
4 protocols using htx multi mode reader
1
Alkaline Phosphatase Activity Assay for Transfected Human MSCs
2
Regulation of Bovine Mammary Epithelial Cell Triglycerides
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The BMECs were transfected with bta-miR-148a-mimic, inhibitor, and shNC in six-well cell culture plates to determine TG contents. After 24 h of successful transfection, the intracellular triglyceride content of BMECs was extracted and detected with the help of a tissue and cell triglyceride assay kit (APPLYGEN, E1013, Beijing, China) following the manufacturer’s protocols. The extracted samples were examined using the software Gen5 CHS (SYNERGY|HTX multi-mode reader, Bio Tek, S1LFTA), and the total TG content was adjusted by the quantity of total protein. Each sample was replicated thrice, and the final results were calculated using the average values. Moreover, this experiment was repeated three times following the same steps mentioned here. The same protocol was used for the intracellular triglyceride content of BMECs transfected with pb7sk-KLF6-siRNA1, pb7sk-GFP-Neo, pBI-CMV3-KLF6, and pBI-GFP-Neo-CMV3.
3
TBARS Assay for Oxidative Stress
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The assay was performed according to the previous report with slight modifications [31 (link)]. Briefly, after pretreatment of cells with PDNE or trolox for 24 h and followed by TBHP for 2 h, the cell culture supernatant was collected and cleared by centrifugation for 5 min, at 10,000 rpm and 4 °C. In a 1.5 mL eppendorf tube, 300 μL of cell-free supernatant was added to 300 μL thiobarbituric acid, TBA (Cayman 10009199) solution (0.5% TBA in 20% trichloroacetic acid and 0.33 M hydrochloric acid solution with 0.005% butylated hydroxytoluene). The eppendorf tubes containing the reaction mixtures were placed in a boiling water bath for 60 min. Subsequently, the samples were placed on ice for 10 min. Then, 300 μL butanol was added to each sample, vortexed and centrifuged at 10,000 rpm for 5 min. From this, 100 μL of the butanol fraction was transferred to 96-well clear-bottom black plates, and fluorescence (excitation, 530 nm, and emission, 590 nm) was read using a Biotek SYNERGY HTX multi-mode reader.
4
Quantifying lipid content in bMECs
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The miR-485 plasmids were transfected into bMECs, and then the TGs, CHOL, and NEFA contents were extracted 36 h later. We used a tissue TG Assay Kit (APPLYGEN, E1013, Beijing, China), Tissue CHOL Assay Kit (APPLYGEN, E1015, Beijing, China), NEFA Detection Kit (Nanjing Jiancheng Bioengineering Institute, A042-2-1, Nanjing, China), and BCA Protein Quantification Kit (Vazyme, E112-02, Nanjing, China) to quantify TGs, CHOL, and NEFA in bMECs. Finally, we used a microplate reader (SYNERGY HTX multi-mode reader, Bio Tek, S1LFTA) to measure the absorbance of the samples.
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