For assay, cells were seeded into 96-well microplates at a density of 5 × 103 cells/well and incubated for 24 h at 37 °C. After medium removal, G3G02N solutions were prepared as described above in medium (200 µL/well). The plates were then incubated for 72 h. Then, plates were centrifuged (2000 rpm, 5 min). Cells were then washed with PBS and fixed in 3.7% formaldehyde solution in PBS and stained with 600 nM DAPI solution in PBS (100 µL/well, 1 h). The fluorescent signal, proportional to the number of cells, was measured in a Tecan Infinite M200 PRO Multimode Microplate Reader (TECAN Group Ltd., Switzerland) at 360/460 nm. The results were expressed as % of the control (DMSO treated cells).
Infinite m200 pro multimode microplate reader
Manufactured by Tecan
Sourced in Switzerland, United States
The Infinite M200 PRO Multimode Microplate Reader is a compact and versatile instrument designed for a wide range of microplate-based applications. It offers a variety of detection modes, including absorbance, fluorescence, and luminescence, allowing researchers to perform diverse assays within a single platform.
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Lab products found in correlation
Pierce bca protein assay, by Thermo Fisher Scientific (10 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Cellevent caspase 3 7 green detection reagent, by Thermo Fisher Scientific (3 mentions)
Dual luciferase reporter assay system, by Promega (3 mentions)
Celltiter glo luminescent cell viability assay, by Promega (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
96 well clear bottom black microplate, by Corning (1 mentions)
Prism 9, by GraphPad (1 mentions)
Millex gp syringe filter unit, by Merck Group (1 mentions)
Cell counting kit 8 (cck8), by Beyotime (1 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Kadcyla, by Roche (1 mentions)
Farmiblastina, by Pfizer (1 mentions)
Human ifn γ elisa max deluxe, by BioLegend (1 mentions)
Protease inhibitors cocktail, by Merck Group (1 mentions)
Prl tk, by Promega (1 mentions)
Nitric oxide colorimetric assay kit, by Abcam (1 mentions)
Bicinchoninic acid reagent, by Thermo Fisher Scientific (1 mentions)
2 7 dichlorofluorescin diacetate dcfda, by Merck Group (1 mentions)
65 protocols using infinite m200 pro multimode microplate reader
1
Cytotoxicity Assay of G3^G02N
2
Quantifying Cell Viability Responses
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Cells in single-cell suspension were plated with culture medium in 96-well clear-bottom black microplates (Cat #3917; Corning Costar, Corning, NY, USA) at a density of 2500 cells per well for adult GBM cell lines (GBM6, GBM 10, GBM14, GBM 39, GBM43, and GBM 108) or 5000 cells per well for DMG cell lines (SU-DIPG XIII-P [47 (link)], SU-DIPG XVII [48 (link)], SF8628, SF8628-B23 (H3F3A K27M knockout of SF8628), and PED17) and cultured overnight at 37 °C with 5% CO2. The next day, cells were treated in triplicate with either vehicle (ddH2O or PBS) or serial dilutions of IL-13 (to final concentrations of 100, 50, 20, 10, 5, 1, and 0.5 ng/mL) or GB-13 (to final concentrations of 320, 100, 32, 10, 3.2, 1, 0.32, 0.1, 0.032, 0.01, 0.0032, and 0.001 ng/mL). Cells were incubated for 72 h and then assayed with CellTiter-Glo Luminescent Cell Viability Assay (Cat #G7570; Promega, Madison, WI, USA) according to the manufacturer’s recommendations. Luminescence was measured using an Infinite M200 PRO multimode microplate reader (Tecan Group, Männedorf, Switzerland), normalized to control wells (ddH2O or PBS only), and relative luminescence treatment was plotted as a function of drug concentration. The potency (50% inhibitory concentration, IC50) of each treatment was calculated by non-linear least-squares curve fitting using Prism 9 (GraphPad, San Diego, CA, USA).
3
Caspase-3/7 Activity Assay for Apoptosis
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Apoptosis was estimated with CellEvent™ Caspase-3/7 Green Detection Reagent (Thermo Fisher Scientific) assay as executive caspases 3 and 7 activity. Cells were seeded in flat-bottom solid black 96-well microplates (2 × 104 or 4 × 104 per well, BJ and SCC-15, respectively) and treated with 1.25–10 µM concentration range of dendrimer conjugates for 24 h. Working solution of CellEvent™ Caspase-3/7 Green Detection Reagent was added (4µ/well) and incubated (1 h, 37 °C). Fluorescence was measured at 490/530 nm with Tecan Infinite M200 PRO Multimode Microplate Reader (TECAN Group Ltd., Switzerland). Results from triplicates of three independent experiments were presented as a percentage of non-treated control.
4
E. coli Stress Resistance Characterization
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The E. coli KNabc wild-type strain and the recombinant strains (E. coli KNabc/pUC19, E. coli KNabc/pET22b, E. coli KNabc/pUC19-AI-2E, and E. coli KNabc/pET22b-AI-2E) were streak cultured on fresh and 100 μg/ml ampicillin-added LBK agar plates, respectively. Single colonies were picked for the expansion. To determine the resistance of the E. coli KNabc strain to alkaline pH, Na+ and Li+; the E. coli KNabc strain and recombinant strains were activated, followed by 1% (v/v) subculture in the LBK medium at different pH (7.0, 7.5, 8.0, and 8.5) and the LBK medium at pH 7.0 but with different concentrations of NaCl (0.2, 0.3, and 0.4 M) or LiCl (5, 10, 25, and 30 mM). After incubation at 37°C for 24 h, the optical density (OD600) values of the bacterial culture were measured by Infinite M200 PRO Multimode Microplate Reader (Tecan Group Ltd., Switzerland). Three replicates were done for each sample.
5
Quantifying AI-2 Signaling in Lactobacillus
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The detection of AI-2 is based on the method of Liu et al. (2018 (link)) with some modifications. Activated L. acidophilus CICC 6074 wild-type strain and recombinant strains (L. acidophilus CICC 6074/pMG36e and L. acidophilus CICC 6074/pMG36e-AI-2E) were added to 12% (w/w) skimmed milk medium without erythromycin. The L. acidophilus cells in cultures at 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 h were removed by centrifugation at 12,000 × g for 10 min at 4°C. The supernatant pH was adjusted to 7.0 and filtered by 0.22 μm Millex-GP Syringe Filter Unit (Merck KGaA, Germany). The obtained supernatant was added to Vibrio harveyi BB170 and the AB medium mixture in a ratio of 1:10 and incubated at 28°C for 5 h. Added 100 μl of culture solution to a 96-well white plate and determined luminescence values using the Infinite M200 PRO Multimode Microplate Reader (Tecan Group Ltd., Switzerland).
6
Salivary Glycoxidation and Nitrosative Stress Assays
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All reagents (unless otherwise stated) were purchased from Sigma-Aldrich Nümbrecht, Germany, and Sigma-Aldrich Saint Louis, MO, USA. Salivary glycoxidation and nitrosative stress assays were performed in duplicate samples. The absorbance/fluorescence was measured using Infinite M200 PRO Multimode Microplate Reader (Tecan Group Ltd., Männedorf, Switzerland). The results were standardized to 1 mg of total protein (TP).
TP content was analyzed using a commercial kit (Thermo Scientific PIERCE BCA Protein Assay; Rockford, IL, USA), according to the manufacturer's instructions. The salivary amylase activity was determined colorimetrically using 3,5-dinitrosalicylic acid (POCH, Gliwice, Poland) as a substrate reaction [40 (link)]. The absorbance was measured at 540 nm.
TP content was analyzed using a commercial kit (Thermo Scientific PIERCE BCA Protein Assay; Rockford, IL, USA), according to the manufacturer's instructions. The salivary amylase activity was determined colorimetrically using 3,5-dinitrosalicylic acid (POCH, Gliwice, Poland) as a substrate reaction [40 (link)]. The absorbance was measured at 540 nm.
7
Redox Assay Protocol for Protein Analysis
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All reagents (unless otherwise stated) were purchased from Sigma-Aldrich Nümbrecht, Germany and Sigma-Aldrich Saint Louis, MO, USA. The absorbance and fluorescence were measured using Infinite M200 PRO Multimode Microplate Reader (Tecan Group Ltd., Männedorf, Switzerland). Redox assays were performed in duplicate samples. Due to the high number of determinations, a detailed description of biochemical methods was given in references. The results were standardized to 1 mg of total protein. The total protein concentration was analyzed using a commercial kit (Thermo Scientific PIERCE BCA Protein Assay; Rockford, IL, USA), according to manufacturer’s instructions.
8
Quantifying Cellular Uptake of G3-BCLF
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Cells were grown in 96-well plates at 2 × 104 cells/well (BJ and U-118 MG) or 4 × 104 (HaCaT). After 24 h, cells were washed with PBS and treated with FITC labeled analog of G3-BCL (G3-BCLF) at 0.1 µM concentration in complete EMEM medium for 1, 3, 6 or 24 h. After incubation cells were fixed in 3.7% formaldehyde and washed with PBS. 600 nM DAPI in PBS was added (100 µL/well) for 1 h at RT. Fluorescence signal was monitored at 485/530 nm for FITC or 360/460 nm for DAPI using Infinite M200 PRO Multimode Microplate Reader (TECAN Group Ltd., Switzerland). The DAPI fluorescence signals were used to estimate number of cells in each well and to calculate fluorescent signal intensity per cell using the constructed calibration curve.
9
Cytotoxicity Assessment of Cell Lines
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The Cell Counting Kit-8 (cat no. C0037; Beyotime Institute of Biotechnology) was used to measure cytotoxicity of both cell lines, according to the manufacturer's instructions. Briefly, the HSC-6 cell line and CAL-27 cell line were seeded into 96-well plates at a cell density of 5×104/ml) overnight. After 24 h, the cell culture medium was replaced by indicated concentrations of chemicals and the treatment was continued for 48 h. CCK8 solution (0.5 mg/ml; 100 µl) was added into each well and incubated for 3 h at 37°C, followed by detection of optical density (OD) values at 450 nm using an Infinite M200 PRO Multimode Microplate Reader (Tecan Group, Ltd.). The percentage of live cells was calculated relative to the control.
10
Quantitative Measurement of Translational Fidelity
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The dual-luciferase reporter plasmids (Supplementary Table S4 ) were used to quantitatively measure −1 programmed ribosomal frameshifting, +1 programmed ribosomal frameshifting, UAA and UAG codon readthrough, and suppression of an AGC serine codon in place of an AGA arginine codon. Cells transformed with either in-frame Renilla-firefly luciferase control or reporter plasmids were grown in synthetic complete medium to exponential growth phase. The cells were collected by centrifugation and resuspended in 1× passive lysis buffer (Promega Corporation, Madison, WI, USA). Luminescence measurements were performed using the Dual-Luciferase Reporter Assay System (Promega) with Infinite M200 Pro Multimode Microplate Reader (Tecan Group Ltd., Männedorf, Switzerland). For each plasmid, at least 15 biological replicates were analysed. For data analysis, the ratio of firefly to Renilla luciferase activity was calculated for each plasmid. The translational fidelity was calculated by dividing the firefly/Renilla ratios determined from cells expressing the reporter plasmids by the firefly/Renilla ratio determined from cells expressing the in-frame control plasmid. The statistical significance was determined by the unpaired two-sample Student’s t-test.
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