The amount of tannins in the SHC extract was determined with slight modifications to the Folin-Ciocalteu method [15 (link)]. 0.5 mL of the SHC extract was added to a brown 25 mL volumetric flask. 0.5 mL of the Folin–Ciocalteu reagent, 2.5 mL of deionized water, and 29% Na2CO3 was used to fix the volume to the scale. The mixture was shaken well and kept at room temperature for 30 min. The reaction mixture absorbance was measured at 760 nm against a deionized water blank on a Multimode Microplate Reader (PerkinElmer Victor Nivo, MA, USA). Gallic acid was chosen as a standard. The correlation equation constructed with gallic acid (0–0.037 mg/mL) was A = 50.723C－0.0615 (R2 = 0.9996). The TTC was expressed as mg of gallic acid equivalents per gram of dry weight. All samples were analyzed in triplicate.
Multimode microplate reader
Manufactured by PerkinElmer
Sourced in United States, Finland
The Multimode Microplate Reader is a versatile instrument that can measure a variety of sample types in a 96-well or 384-well microplate format. It is capable of multiple detection modes, including absorbance, fluorescence, and luminescence, allowing for diverse analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Cell counting kit 8 (cck8), by Dojindo Laboratories (4 mentions)
Luciferase substrate, by Promega (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Total glutathione assay kit, by Beyotime (1 mentions)
Polyethyleninime, by Polysciences (1 mentions)
Gag pol expression plasmid, by Addgene (1 mentions)
Slhp033rb, by Merck Group (1 mentions)
Cytokine elisa kits, by BioLegend (1 mentions)
Luciferase kit, by Promega (1 mentions)
Lysis reagent, by Promega (1 mentions)
Reactive oxygen species assay kit, by Beyotime (1 mentions)
Corresponding detection kit, by Nanjing Jiancheng (1 mentions)
Ldh cytotoxicity assay kit, by Promega (1 mentions)
Recombinant human insulin, by PromoCell (1 mentions)
Wst 1 reagent, by Roche (1 mentions)
28 protocols using multimode microplate reader
1
Tannin Content Determination in SHC Extract
2
Proliferation of HaCaT and CCD-986sk Cells
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The proliferation capacities of HaCaT and CCD-986sk cells used to evaluate the biological activities of the LMWP-GFs, QCN, and OXY-PFOB-NE were assessed. Briefly, HaCaT and CCD-986sk cells were seeded at a density of 5×103 cells/well and 1×104 cells/well, respectively, in 200 µL of Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in 96-well plates. Afterward, the HaCaT and CCD-986sk cells were cultured at 37°C in 95% air and 5% CO2 for 24 h and 48 h, respectively. After overnight incubation in serum-free medium, the cells were treated for 24 h in low-serum medium (0.05% FBS) with LMWP-GFs comprising LMWP-EGF (500 ng/mL), LMWP-IGF-I (500 ng/mL), LMWP-PDGF-A (10 ng/mL), and LMWP-bFGF (10 ng/mL), QCN in 10% DMSO (0.1–25 µg/mL), and an aqueous dispersion of OXY-PFOB-NE (0.03–300 µg/mL PFOB-NE) alone or combined. Cell proliferation was assessed by adding 10 µL of WST-1 reagent to each well and incubating at 37°C for 1 h. Absorbance was measured at 450 nm using a multimode microplate reader (PerkinElmer, Waltham, MA, USA). The percentage of viable cells was calculated by comparing the values of treated cells to those of untreated cells for both the HaCaT and CCD-986sk cells.
3
Dual-Luciferase Assay of SIRT7-miR-130a-3p Interaction
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TargetScan (http://www.targetscan.org ) online tool was utilized to predict which gave a prediction that miR‐130a‐3p can bind to the 3’UTR of SIRT7. SIRT7 wild‐type vectors, SIRT7 mutant‐type vectors, miR‐130a‐3p vectors and blank vectors were purchased from Genepharma (China). For dual‐luciferase reporter gene assay, HEK293T cells were transiently transfected with SIRT7 wild‐type or SIRT7 mutant‐type plasmids, and miR‐130a‐3p plasmids using Lipofectamine 2000 (Invitrogen). Report gene assay was performed 24 hours after transfection using Dual‐Luciferase Reporter Assay System (Promega, USA). Firefly Luciferase and Ranilla Luciferase were detected by multi‐mode microplate reader (PerkinElmer, USA). Firefly/Ranilla Luciferase activity was used for internal control, and all assays were repeated three times.
4
Aluminum Chloride Colorimetric Assay for Total Flavonoid Content
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The TFC was determined according to the aluminum chloride colorimetric assay [14 (link)]. 0.2 mL of the solution was added to a 10 mL volumetric flask, and mixed with 0.3 mL of 5% NaNO2 for 6 min. 0.3 mL of 10% AlCl3 was added, shaken well and left for another 6 min. The reaction stopped when it reached 4 mL of 4% NaOH and deionized water was used to fix the volume to the scale. After being incubated at room temperature for 15 min, the reaction mixture absorbance was measured at 514 nm against a deionized water blank on a Multimode Microplate Reader (PerkinElmer Victor Nivo, MA, USA). Rutin was chosen as a standard. The correlation equation constructed with rutin (0–0.016 mg/mL) was A = 6.2621C-0.0027 (R2 = 0.9997). The TFC was expressed as mg of rutin equivalents per gram of dry weight. All samples were analyzed in triplicate.
5
SARS-CoV-2 Pseudovirus Neutralization Assay
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The pseudovirus neutralization was shown in a previous study.47 (link) Briefly, immune sera were gradiently diluted by DMEM with 10% fetal bovine serum and then pre-incubated with EGFP-expressing or luciferase-expressing pseudoviruses in 96-well plates for 1 h at 37 °C, including both WT and variant (B.1.1.7, B.1.351, P.1, P.1.617.2, C.37, and Omicron) pseudoviruses. Then add 293 T/ACE2 cells at a density of 1 × 104 cells per well and incubate for an additional 48 h for expression.
The efficiency of viral entry was tested with a firefly luciferase assay. In brief, the supernatants of infected cells were removed and 50 μl PBS and 50 μl lysis reagent from a luciferase kit were added. Then add luciferase substrates (Promega) to wells and detect relative light units with a multi-mode microplate reader (PerkinElmer). Use fluorescent microscopy and FCM to determine the infection efficiency of EGFP-expressing pseudoviruses in 293T/ACE2 cells.
The efficiency of viral entry was tested with a firefly luciferase assay. In brief, the supernatants of infected cells were removed and 50 μl PBS and 50 μl lysis reagent from a luciferase kit were added. Then add luciferase substrates (Promega) to wells and detect relative light units with a multi-mode microplate reader (PerkinElmer). Use fluorescent microscopy and FCM to determine the infection efficiency of EGFP-expressing pseudoviruses in 293T/ACE2 cells.
6
Quantifying Cellular Glutathione Levels
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Cellular GSH levels were measured using the Total Glutathione Assay Kit (Beyotime Biotechnology, S0052). Briefly, cells were washed with PBS and protein removal reagent S solution was added. Later, the cells were quickly lysed via freeze/thawing twice and were centrifuged at 10,000×g for 10 min at 4 °C according to the manufacturer’s instructions. The supernatant was mixed with 2 mM 5,5′-dithio-bis (2-nitrobenzoic acid), Glutathione reductase, and NADPH. The GSH levels were measured via the glutathione reductase recycle assay. The optical density at 412 nm was read using a multimode microplate reader (PerkinElmer, USA) and the GSH concentrations were calculated against a standard curve.
7
SARS-CoV-2 Pseudovirus Neutralization Assay
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We purchased variants of SARS‐CoV‐2 pseudoviruses (GFP Luciferase) from Genomeditech Company (China) except BA.3 and BA.4/5 pseudoviruses, which were bought from the Vazyme company (China). NIH mice were intramuscularly injected with mRNA vaccine. After being inactivated at 60°C for 30 min, sera were diluted by a triple‐gradient by complete DME medium. And then, diluted sera were incubated with luciferase‐expressing pseudovirus (prototypes, B.1.1.7, B.1.351, P.1, B.1.617, C.37, BA.1, BA.2, BA.2.12.1, BA.3, and BA.4/5) at 37°C for 1 h. 1.2 × 104 293T/ACE2 cells were added to each well to express the reporter gene. A period of 48 h later, after removing the infected cell supernatants, 100 µL lysis reagents with luciferase substrate were added to each well. Finally, the plates were detected by a multimode microplate reader (PerkinElmer, USA).
8
SARS-CoV-2 Spike Pseudovirus Generation
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Pseudovirus with spike proteins of SARS-CoV-2 was generated as described previously [31 (link)]. Briefly, 293T cells were transfected with a plasmid encoding codon-optimized SARS-CoV-2 spike protein with a C-terminal 18 aa truncation, a lentiviral vector carrying either EGFP (for FACS assay) or Luc2 (for luciferase assay) reporter, and a gag/pol expression plasmid (Addgene, 12260) using polyethyleninime (Polyscience). Six hours post transfection, the medium was replaced with new complete culture medium. Forty-eight hours post infection, the culture supernatants containing pseudovirus were harvested, filtered through a 0.45 μM pore-size filter (Millipore, SLHP033RB), subjected to ultracentrifugation, and stored at −80 °C prior to infection assays. EGFP or luciferase expression in infected 293 T/ACE2 cells was determined by fluorescence microscopy, flow cytometry or a multimode microplate reader (PerkinElmer).
9
Cell Viability Assay Protocol
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Cells were seeded in a 96-well plate as a density of 5000 cells/well for 24 h. Cell viability was measured using the Cell Counting Kit-8 (Dojindo Molecular Technologies, Japan), according to the manufacturer’s instructions. Absorbance was determined using the Multi-Mode Microplate Reader (PerkinElmer, Finland).
10
SARS-CoV-2 Pseudovirus Neutralization Assay
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The wild type of SARS-CoV-2 luciferase-expressing pseudoviruses was purchased from Genomeditech, China. The pseudovirus neutralization was performed as described previously.16 (link) Briefly, luciferase-expressing pseudovirus was pre-incubated with serially diluted immune sera in 96-well plates for 1 h at 37 °C, following adding the mixture to 293 T/ACE2 cells and then incubating for 48 h to express the reporter gene. The efficiency of viral entry was determined with a firefly luciferase assay. In brief, remove the supernatants of infected cells, then add 50 µl of PBS, 50 µl of lysis reagent from a luciferase kit and luciferase substrate (Promega, USA). Relative light units were performed using a multi-mode microplate reader (PerkinElmer, USA).
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