Bacterial membrane depolarization was measured using a membrane potential-sensitive fluorescent dye, DiSC3-5 (Sigma–Aldrich, Shanghai, China), that changes its fluorescence intensity in response to changes in transmembrane potential. Midlog-phase bacterial cells were centrifuged at 3000 × g for 10 min, washed twice with 5 mM HEPES (pH 7.2) containing 20 mM glucose and 100 mM KCl, and resuspended in the same solution at a final concentration of 2 × 106 CFU/ml. DiSC3-5 was added to a final concentration of 4 μM in a black NBS microplate, and the change in dye fluorescence was continuously monitored at an excitation wavelength of 622 nm and an emission wavelength of 670 nm at 30 s intervals using a Varioskan Flash Microplate Reader (Thermo Scientific Co., Beijing, China). When a stable reduction in the fluorescence occurred because of quenching of the accumulated dye in the membrane interior, DiSC3-5 had achieved maximal uptake by the bacteria. The peptides at a final concentration of 1/4, 1, or 4 times their MIC; 3.125 μM penicillin (1/32 × MIC); and the combination of peptides (1/4 × MIC) and 3.125 μM penicillin were added, and the membrane potential was determined by the change in fluorescence intensity. Triton X-100 was used as a control reference. The measurements were repeated at least three times for each dye concentration.
Flash microplate reader
Manufactured by Thermo Fisher Scientific
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The Flash microplate reader is a compact and versatile instrument designed for rapid fluorescence, luminescence, and absorbance measurements in microplate format. It features high-performance optics and sensitive detectors to provide accurate and reliable data.
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Lab products found in correlation
Disc3 5, by Thermo Fisher Scientific (1 mentions)
Disc3 5, by Merck Group (1 mentions)
Pmirglo dual luciferase mirna target expression vector, by Promega (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
Celltiter glo luminescent cell viability assay kit, by Promega (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
11 protocols using flash microplate reader
1
Bacterial Membrane Potential Depolarization
2
Microplate BCA Protein Assay
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The total cellular protein was determined by using a microplate BCA protein assay kit (KangWei, China). Aqueous BSA standards were prepared from a 2 mg/mL BSA stock solution by serial dilutions. Ten microliters of diluted cell lysate samples or BSA standards were dispensed into a 96-well plate and then 200 μL of the BCA reagent were added. The mixture was incubated at 37 °C for 30 min before reading the absorbance at 595 nm on a Thermo Varioskan Flash microplate reader (Winooski, VT, USA). The protein concentration of each sample was determined by plotting against the standard curve.
3
AuNP-Based Colorimetric Detection
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After the PCR reaction, the product (10 μL) was mixed with 90 μL of labelled AuNps solution and incubated at room temperature for 10 min. Then the mixture was ultra-sonicated for 10 s, and 2 μL of 1 M MgCl2 was added. The color changes of solutions were observed by the naked eyes and photographed by a digital camera. Finally, the absorption spectra were recorded with a Thermo Varioskan Flash Microplate Reader in 20 min.
4
ATG5 3'UTR Luciferase Assay
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The 3′UTR of ATG5 was amplified by PCR and cloned into pmirGLO Dual-Luciferase miRNA target expression vector (Promega, Madison, WI).The predicted target site was mutated by site-directed mutagenesis. For luciferase reporter assays, wild type and mutated luciferase plasmid and agomiR/antagomiR were cotransfected into Hela cells. Luciferase activities were measured using the Dual-Glo® Luciferase Assay System (Promega, Madison, WI, USA) on a Varioskan Flash microplate reader (Thermo Scientific, MA, USA) according to the manufacturer’s protocols. Firefly luciferase activity was normalized to Renilla luciferase activity for analysis. Each experiment was repeated in triplicate.
5
Antioxidant Activity Evaluation of β-carotene Emulsion
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The β-carotene linoleic acid model system was performed as previously described by Wu et al. [1] (link), with some modifications. 2 mg of β-carotene was mixed with 20 mg of linoleic acid and 200 mg of Tween 40 in 1 mL of chloroform. After mixing vigorously, chloroform was completely evaporated under vacuum, and 50 mL of oxygenated distilled water was added. Gently shaking the mixture, a clear yellowish emulsion was formed and freshly prepared before each experiment. 30 μL of R. typhina L. extract was mixed with 250 μL of the β-carotene and linoleic acid emulsion. After sufficient and rapid mixing, the absorbance values of the reaction mixtures were measured at 470 nm immediately and then measured after 120 min at 50 °C on a Varioskan Flash microplate reader (Thermo Scientific, USA). The negative control was methanol while butylated hydroxytoluene (BHT) was included as a positive control. The antioxidant activity (AA) of the samples was calculated by using the following equation: AA (%) = (A2 hsample - A2 hcontrol / A0 hsample - A0 hcontrol) × 100. Extract concentration providing IC50 value was defined by plotting the inhibition percentage versus the extract concentrations.
6
Liver-on-Chip Biomarker Secretion
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Cell morphology and metabolic activity were analyzed via microscopy and ELISA assays to determine the secretion rate of hepatic biomarkers in the chip in flow-through media.
The flow-through fractions obtained over 24 h of the experimental period were analyzed for albumin and urea secretion rates using ELISA according to the Invitrogen manual. All samples were analyzed in duplicates. For the urea detection, to account for the phenol red readouts in DMEM, the absorbance values of the DMEM control were subtracted from the data points. The plates were read at 450 nm on a Varioskan Flash microplate reader (Thermo Scientific, Waltham, MA, USA) for both assays.
Cell viability was confirmed at the end of the flow experiment after the samples were stained with a live/dead staining kit based on Calcein-AM and Propidium Iodide, according to the protocol provided by Sigma-Aldrich.
The flow-through fractions obtained over 24 h of the experimental period were analyzed for albumin and urea secretion rates using ELISA according to the Invitrogen manual. All samples were analyzed in duplicates. For the urea detection, to account for the phenol red readouts in DMEM, the absorbance values of the DMEM control were subtracted from the data points. The plates were read at 450 nm on a Varioskan Flash microplate reader (Thermo Scientific, Waltham, MA, USA) for both assays.
Cell viability was confirmed at the end of the flow experiment after the samples were stained with a live/dead staining kit based on Calcein-AM and Propidium Iodide, according to the protocol provided by Sigma-Aldrich.
7
Stable Transfection Cell Proliferation Assay
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For the stable transfection cell proliferation assay, A498, A498-Con, A498-Tiki2 cells were seeded at a density of 2 × 103 per well in 96-well microplates and incubated for 96 h. Cell viability was determined every 24 h using a CellTiter-Glo luminescent cell viability assay kit (Promega, USA), and measured by Varioskan Flash microplate reader (Thermo, USA) according to the manufacturer's protocol. Each experiment was repeated independently at least three times in triplicate. For the cell proliferation assays with TIKI2 siRNA, the 769-P cells were seeded at a density of 2 × 103 per well in 96-well microplates the day before the transfection. After the transient transfection, the cells were incubated for 96 h using the same method as described above.
8
Quantifying Recombinant Human Acidic Fibroblast Growth Factor in Tissues
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A double-sandwich ELISA was developed to measure the concentration of rh-aFGF in skin tissue homogenates and sera from rats. In brief, a 96-well microplate was pre-coated with anti-human rh-aFGF antibody (150 μL/well). An rh-aFGF quality control (QC) sample or test sample (50 μL/well) was added, and the wells were incubated at room temperature for 2 h with shaking. The liquid in the well was discarded, and wells were washed four times with washing buffer (300 μL/well). Then, an enzyme-labeled antibody (200 μL/well) was added. After incubation at room temperature for 2 h with shaking, the liquid in the well was discarded, and the washing buffer (300 μL/well) was added. The plate was washed four times, tetramethyl benzidine (TMB) substrate (200 μL/well) was added, and the plate was incubated at room temperature in the dark for 30 min. The reaction was stopped with the addition of 2 M of H2SO4 stop solution (50 μL/well). Absorbance (450 nm) was measured with a Varioskan Flash microplate reader (Thermo, USA), with a reference setting at 570 nm.
9
Cytotoxicity Evaluation of MT-1 on HeLa Cells
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The cellular cytotoxicity of MT-1 toward HeLa cells was evaluated using the standard methyl thiazolyl tetrazolium (MTT) assay. HeLa cells were seeded into a 96-well plate at a concentration of 4 × 103 cells/well in 100 μL of dulbecco’s modified eagle medium (DMEM) medium with 10% fetal bovine serum (FBS). The plate was maintained at 37 °C in a 5% CO2, 95% air incubator for 24 h. After the original medium was removed, the HeLa cells were incubated with different concentrations of MT-1 (from the stock solution in Section 2.3 with different volumes, final concentrations: 1, 2 and 5 μM, containing DMSO as cosolvent, all final DMSO concentrations were adjusted to 1% with culture solvent, V/V) for 24 h. The cells only incubated with the culture solvent (containing 1% DMSO as cosolvent) served as the control. The cells were washed with PBS three times, and then 100 μL MTT solution (0.5 mg/mL in PBS) was added to each well and incubated for 4 h. Then the supernatant was removed gently. After the addition of DMSO (150 μL/well), the assay plate was shaken at room temperature for 10 min. The spectrophotometric absorbance of the samples was measured by using a Thermo Fisher-Varioska Flash microplate reader for optical density at 490 nm.
10
Quantifying Recombinant Human aFGF in Skin
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In order to eliminate interference from endogenous aFGF and related proteins, we first investigated the effects of the dilution ratio of skin homogenates on the accuracy of rh-aFGF measurements. For this purpose, we used RD6X buffer to prepare a dilution gradient of skin homogenates at 1:2.5, 1:5, 1:10, and 1:20. Absorbance (450 nm) was measured with a Varioskan Flash microplate reader (Thermo, USA), with a reference setting at 570 nm.
Two different biological matrices (RD6X buffer and 10× diluted skin homogenate) were selected to prepare the calibration curve (62.5, 125, 250, 500, 1,000, 2,000, and 4,000 pg/mL). rh-aFGF QC samples and rh-aFGF test samples were diluted to three concentrations (160, 800, and 3,200 pg/mL), and the concentrations were determined with the calibration curve.
Two different biological matrices (RD6X buffer and 10× diluted skin homogenate) were selected to prepare the calibration curve (62.5, 125, 250, 500, 1,000, 2,000, and 4,000 pg/mL). rh-aFGF QC samples and rh-aFGF test samples were diluted to three concentrations (160, 800, and 3,200 pg/mL), and the concentrations were determined with the calibration curve.
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