The whole sequence of circZKSCAN1 and mutated circZKSCAN1 start codon was cloned into PL05-ciR (GENESEED, China) vector. Specific shRNAs for circZKSCAN1 designed to target the covalently closed junction was amplified into PLKO.1-TRC plasmid (Tsingke, China). The whole sequence of circZKSaa and circZKSaa with mutated circZKSaa start codon was amplified into pLV-(2A)-puro vector (Tsingke, China). The whole sequence of mTOR, FBXW7 and mutated mTOR was amplified into pCDN3.1(+) or pCMV-HA vector (Tsingke, China). The two potential IRES sequences of circZKSaa were cloned into Psi-check2 vector (Promega, USA). Both plamids were transfected using LIPO 3.0 (HANBIO, China) at a final concentration of 20 nM. Transfected cells were harvested for indicated analyses 2 d after transfection. Dual luciferase reporter assay used Hela cells, luminescent reaction solution (Galen, China), multi-function microplate reader (Tecan, China). The firefly luciferase readings were measured and the corrected relative luciferase activity was obtained by dividing the desired fluorescence value according to the vector characteristics.
Multifunction microplate reader
Manufactured by Tecan
Sourced in Switzerland, China
The Multifunction Microplate Reader is a versatile instrument designed for the simultaneous detection and quantification of multiple analytes in a microplate format. It offers a range of detection modes, including absorbance, fluorescence, and luminescence, enabling comprehensive sample analysis across various application areas.
Automatically generated - may contain errors
9 protocols using multifunction microplate reader
1
Cloning and Characterization of circZKSCAN1 and circZKSaa
2
Fengycin Effects on A. solani ATP
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The same design as described above was used to investigate the effects of fengycins on the mycelial ATP contents (Wang et al., 2020 (link)). For A. solani, 5-mm2 plugs of mycelial agar were immersed in an MIC value dose of fengycins for 20, 40, 60, 80, 100, and 120 min at 25°C. Plates without fengycins were used as controls. The A. solani cells and the supernatants were collected by centrifugation (12,000 × g, 5 min, 4°C) independently. The extracellular ATP level was determined using an Enhanced ATP Assay Kit (Beyotime Biotechnology Inc., Shanghai, China) and a multi-function microplate reader (Tecan Spark, Salzburg, Austria). The ATP kit was based on a luminescent ATP assay protocol that involves the lysis of the cell samples, addition of the luciferase enzyme and luciferin, and measurement of the emitted light. The experiment was repeated in triplicate.
3
Cell Proliferation Assay Protocol
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Cells were seeded in wells (1×104 cells/well) and cultured for five days. Optical density was tested with the multifunction microplate reader (Tecan, Mannedorf, Switzerland). The assays were performed in triplicate, with at least eight replicates per experiment.
4
Cell Proliferation Assay Protocol
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Cells of passages 3 or 5 were seeded on 96-well microplates with a density of 1000, 2000, 4000, 8000, 16000, or 32000 cells/well. After incubating at 37°C for 4 h, the media were discarded and 110 μL CCK-8 solution (DMEM/F12 without phenol (Gibco, 11039-021): CCK-8 (MCE, HY-K0301) = 10:1) was added into each well. After 2 h, OD at 450 nm was measured using a multifunction microplate reader (Tecan). In a parallel experiment, cells of passages 3 or 5 were plated in 96-well microplates at a density of 1×104 cells/well. The cells were counted each day until the 8th day. Growth curves were plotted using the mean values, and the population doubling time was calculated from the growth curve.
5
Fungal ATP Response to 6-Methyl-2-Heptanone
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The same design as described above was used to investigate the effects of 6-methyl-2-heptanone on outer mycelial ATP contents. For A. solani, a 5-mm square plug of mycelial agar disks was placed in one compartment of the divided plate containing PDA medium, and the plate was incubated at 25°C for 4 days. Then, an EC50 dose of 6-methyl-2-heptanone was added to the other compartment and incubated for 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 h at 25°C. The plates without 6-methyl-2-heptanone were used as controls. The A. solani cells and the supernatants were collected by centrifugation (12,000 × g for 5 min at 4°C). The extracellular ATP level was determined using an Enhanced ATP Assay Kit (Beyotime Biotechnology Inc., Shanghai, China) and a multi-function microplate reader (Tecan Spark, Salzburg, Austria). The ATP kit was based on a luminescent ATP assay protocol that involved the lysis of each cell sample and the addition of luciferase and luciferin, followed by the measurement of the emitted light. The experiment was repeated in triplicate.
6
Cell Proliferation Assessment via CCK-8
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Cell proliferation ability was performed using CCK-8 analysis. In accordance with the CCK-8 kit instructions (Vazyme, Nanjing, China), the specific steps were as follows: embryonic myoblasts were seeded into the 96-well plates cultured in GM, and were transfected for 24–48 h, with six replicates per group. A 10 μL of cck-8 solution was added into each well gently to avoid bubbles, and cells were incubated at 37 °C in darkness for 2–3 h. Based on the fact that WST-8 could be reduced to highly water-soluble orange methylene dye (formazan) by some dehydrogenases in mitochondria under the action of electron coupling carrier 1-Methoxy PMS, the amount of formazan is proportional to the number of living cells. The optical density (OD) value at 450 nm was detected using a multifunction microplate reader (Tecan, Männedorf, Switzerland) at 0 h, 24 h, 48 h, and 72 h, which could indirectly reflect the relative cellular activity.
7
Assessing Cytotoxicity of GS-Rb1 on H9c2 Cells
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Cytotoxicity was assessed using a Cell Counting Kit-8 (CCK8; Beyotime, Shanghai, China) [9 (link)]. GS-Rb1 (≥98%, Solarbio, Beijing, China) was dissolved in serum-free DMEM/F12 and filtered through a 0.22 μm filter. H9c2 cells were seeded into 96-well plates at a density of 1 × 104 per well, and GS-Rb1 was added after 12 hours of culture at final concentrations of 50 μM, 100 μM, 150 μM, and 200 μM, respectively. Controls received serum-free DMEM/F12 without GS-Rb1. After 6 h, 12 h, or 18 h, the medium was removed, and 10 μl CCK-8 solution and 100 μl medium were added to each well for 1 hour. Next, the OD value at 450 nm of each well was read by a multifunction microplate reader (TECAN, Switzerland). Blank values from wells containing only DMEM/F12 without cells were subtracted. The cell viability of the 0 μM group was set at 100%, and the relative cell viability of the other groups was calculated. OD values were determined for three replicate wells for each experimental point.
8
Measuring cAMP Levels in GBM Cells
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GBM cells were treated with DMSO or CG500354 (3 μM/ml) for 72 hours. The cAMP level was measured from the collected supernatants using a cyclic AMP EIA Kit (#581001, Cayman Chemical Company, Ann Arbor, MI, USA) according to the manufacturer's instructions. The cAMP values were calculated using a multifunction microplate reader (Tecan, San Jose, CA, USA).
9
Cell Viability and Colony Formation Assay
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1.0 × 104 cells were plated in 96-well plates to assess cell viabilities. Different concentrations of NaHS (0, 10, 25, 50, 100, 200 μM) and H2O2 (0, 20, 40, 60, 80, 100, 200 mM) were added to the serum-free medium. After 12 h treatment, cells were assessed via an MTT assay and the optical density was measured at 490 nm by a multifunction microplate reader (Tecan Infinite, Mannedorf, Switzerland).
3 × 102 A549 cells/well were seeded in 6-well plates for a colony formation assay. After two weeks, colonies were fixed in methanol, stained with 0.1% crystal violet, and photographed to count the number.
3 × 102 A549 cells/well were seeded in 6-well plates for a colony formation assay. After two weeks, colonies were fixed in methanol, stained with 0.1% crystal violet, and photographed to count the number.
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