The total phenolic content (TPC) of the extracts was calculated using the Folin–Ciocalteu reagent method [16 (link)]. We added 500 μL of the sample or standard, diluted with 50 μL Folin–Ciocalteau reagent (1 N; Sigma-Aldrich), and left it to stand for 3 min. Next, 100 μL of 20% Na2CO3 was added to the mixture, which was then shaken and incubated at room temperature for 60 min. For the analysis of the total phenol content, Folin–Ciocalteau reagent, which colored blue when reacted with phosphomolybdic acid, was used, and the value was expressed as gallic acid equivalent (mg GAE/g). All samples were analyzed in 3 repetitions, and absorbance was measured at 732 nm using a multi-plate reader (BioTek Instruments, Winooski, VT, USA). The total flavonoid content (TFC) in the extracts was determined using the Folin–Ciocalteu reagent method [16 (link)], with some modifications. A total of 150 μL of the solution (standard or CLF1 and CLF2) was mixed with 0.3 mL of NaNO2 (5%, w/v). After 5 min, 10 μL of AlCl3 (10%, w/v) was added. The sample was mixed and neutralized after 6 min with 0.5 mL of 1 M NaOH solution. The mixture was then left for 10 min at room temperature. The catechin equivalent (mg of CAE/g) was the TFC standard, and absorbance was detected at 510 nm using a multi-plate reader (BioTek Instruments).
Multiplate reader
Manufactured by Agilent Technologies
Sourced in United States
The Multiplate reader is a versatile laboratory instrument designed for performing a variety of microplate-based assays. It provides accurate and reliable results for applications such as colorimetric, fluorometric, and luminometric measurements. The device offers a flexible and user-friendly platform for researchers and scientists across various fields of study.
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Lab products found in correlation
Luciferase reporter assay system, by Promega (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Cck 8 reagent, by Beyotime (2 mentions)
Cell count kit 8 assay, by Beyotime (2 mentions)
Originpro 8, by OriginLab (2 mentions)
Cell counting kit 8 (cck8), by Beyotime (1 mentions)
Cignal finder 96 well plates, by Qiagen (1 mentions)
Cck 8, by Dojindo Laboratories (1 mentions)
Fetal bovine serum (fbs), by ExCell Bio (1 mentions)
Mcp 1, by Merck Group (1 mentions)
Vegf a, by R&D Systems (1 mentions)
Cytotox glo cytotoxicity assay, by Promega (1 mentions)
Cck 8, by Beyotime (1 mentions)
Cell counting kit 8 (cck8), by Thermo Fisher Scientific (1 mentions)
Venetoclax, by Selleck Chemicals (1 mentions)
96 well plate, by Thermo Fisher Scientific (1 mentions)
Palbociclib, by Selleck Chemicals (1 mentions)
Prism 6, by GraphPad (1 mentions)
Pgp glotm assay system, by Promega (1 mentions)
Alexa fluor 488 protein labeling kit, by Thermo Fisher Scientific (1 mentions)
59 protocols using multiplate reader
1
Quantifying Total Phenolic and Flavonoid Content
2
Cell Proliferation Quantification via CCK-8 Assay
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Cell proliferation was quantified through the utilization of Cell Counting Kit-8 assays (CCK-8, Beyotime). Cells were seeded into 96-well plates at a density of 1 × 103 cells per well and treated with the leachate from each group. At predetermined intervals (1, 3, 5, and 7 days), 100 μl of the CCK-8 solution was added and the cells were incubated for 1 h at 37°C. Optical density (OD) was subsequently measured at a wavelength of 450 nm using a multiplate reader (BioTek, Winooski, VT, USA).
3
Screening Signaling Pathways in Tenogenesis
4
Cell Growth Monitoring with CCK-8 Assay
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Cell counting kit-8(CCK-8, Dojindo) was used to assay cell growth . 1× 103 cells were grown in 96-well plates for 24hr through 144 hours. Every 24 h term, cell growth was detected using a CCK-8 assay kit followed by company instruct protocol. 10 μl of CCK-8 solution was added to each well, followed by incubation for 3 h at 37°C. The absorbance at 450 nm was determined by a multiplate reader (BioTek). The mean value and STDEV from 4 wells were calculated.
5
Murine Macrophage Response to Phytochemicals
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Murine macrophage cell line RAW 264.7 were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Hyclone Florida, USA) supplemented with 10% fetal bovine serum (FBS, ExCell Bio Shanghai, China), 1% Penicillin Streptomycin (Gibico California, USA) at 37 °C in a humidified atmosphere of 95% air and 5% CO2. After spreading at 80–90% confluence, cells were washed with PBS, scraped with fresh culture, and subcultured into 96-well plates at a density of 5.0 × 103 cells/well and incubated with or without LPS (1 μg/mL). The cells were exposed to different concentrations of gentiopicroside, loganic acid, swertiamarin, and vitexin (0, 1, 5, 10, 20, 40, and 50 μM) with or without LPS (1 μg/mL) for 24 h. The optical density was measured at 450 nm using a multi-plate reader (BioTek, Winooski, VT, USA).
6
Cell Viability Assay via DDX21 Knockdown
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A549 and H1299 cells were infected with 513B-DDX21 virus or transfected with siRNA targeting DDX21 for 24 h, and then cells were seeded in 96-well plates (5 × 103 cells/well) and cultured in an incubator for 0, 24, 48, or 72 h. After discarding the medium, 100 μl medium and 10 μl CCK-8 (VICMED, VC5001L-2500) were mixed in a 1.5 ml EP tube, and the prepared CCK-8 solution was added to the culture plate. After incubation at 37°C for 1 h, the optical density at 450 nm (OD450) was measured by a multi-plate reader (BioTek, USA).
7
Astrocyte Cytokine Profiling by ELISA
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The cell culture supernatants of astrocytes subjected to different treatments were harvested and assayed with Enzyme-linked immunosorbent assays (ELISAs) to determine the levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, transforming growth factor-β (TGF-β), CXCL-1, VEGF-A (R&D Systems, USA), and MCP-1 (Sigma-Aldrich, USA) according to the manufacturer’s protocols. The results were calculated by measuring the absorbance at a wavelength of 450 nm using a multiplate reader (Bio-Tek, Winooski, VT, USA). The experiments were performed in triplicate.
8
Cytotoxicity of 2-DG on Human Astrocytes
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To determine the cytotoxicity of 2-DG on human astrocytes, cells were cultured in 96-well white tissue culture plates and treated as described above (Section 2.2 ) and analyzed for cytotoxicity using the CytoTox-Glo Cytotoxicity Assay (Promega, Madison, WI, USA, cat. no. G9290) as per the manufacturer’s instructions. Luminescence was measured using a BioTek multiplate reader. A positive control (lysis buffer) for cell death was used to calculate the percent cell death in each well and quantities compared between treatment groups. Experiments were performed in triplicate and repeated three times.
9
Cell Proliferation Assay Using CCK-8
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The cell proliferation was assessed by CCK-8 (Beyotime, China) following the manufacturer’s instructions. Briefly, cells were seeded on a 96-well plate in triplicates at a density of 1000 cells/200 μl. The medium in each well was replaced with 100 μl of fresh medium supplemented with 10 μl of CCK-8 reagent. At indicated time points and incubated at 37 °C for 3–4 h. The optical density (absorbance) at a wavelength of 450 nm was determined by a multiplate reader (Bio Tek, Vermont, USA).
10
Quantifying Cell Colony Formation and Viability
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For the colony formation assay, cells were seeded into 12-well plates at a density of 1000 cells per well. The cells in the plates were incubated for approximately 7-10 days in fresh medium, washed with PBS, fixed with 95% ethanol and stained with 0.1% crystal violet. Ultimately, the numbers of cell colonies representing colony-forming ability were imaged.
For the CCK8 assay, 1x103 HB cells with the indicated transfection were seeded in triplicate into 96-well plates containing 200 μl culture medium in each well. At the indicated time points, the medium of each well was replaced with 100 μl fresh medium supplemented with 10 μl CCK8 reagent (Beyotime, Jiangsu, China), and the cells were incubated for another 2h. Finally, a multiplate reader (Bio Tek, Vermont, USA) was used to determine the absorbance at a wavelength of 450nm.
For the CCK8 assay, 1x103 HB cells with the indicated transfection were seeded in triplicate into 96-well plates containing 200 μl culture medium in each well. At the indicated time points, the medium of each well was replaced with 100 μl fresh medium supplemented with 10 μl CCK8 reagent (Beyotime, Jiangsu, China), and the cells were incubated for another 2h. Finally, a multiplate reader (Bio Tek, Vermont, USA) was used to determine the absorbance at a wavelength of 450nm.
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