Cell proliferation was measured with BrdU incorporation assay using the chemiluminescent BrdU Cell Proliferation Assay Kit (Roche) as it was performed [60 (link)]. Chemiluminescence was measured using the Tecan Spectra Fluor Plus Microplate Reader (Tecan Group Ltd.) in three independent experiments, each performed in triplicate.
Spectrafluor plus microplate reader
Manufactured by Tecan
Sourced in Switzerland, Austria, Germany
The SpectraFluor Plus Microplate Reader is a versatile instrument designed for fluorescence and absorbance measurements in microplates. It is capable of detecting a wide range of fluorescent and colorimetric assays, making it suitable for various applications in life science research and clinical diagnostics.
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Lab products found in correlation
Duoset elisa assay, by R&D Systems (2 mentions)
Mtt colorimetric assay, by Merck Group (1 mentions)
Atplite luminescence atp detection assay system, by PerkinElmer (1 mentions)
Brdu cell proliferation assay kit, by Roche (1 mentions)
Mtt solution, by Merck Group (1 mentions)
Celltiter glo luminescent cell viability kit, by Promega (1 mentions)
Oil red, by Merck Group (1 mentions)
Psicheck 2 vector, by Promega (1 mentions)
Dual glo luciferase assay kit, by Promega (1 mentions)
Ultrasensitive mouse insulin elisa, by Mercodia (1 mentions)
Tubulin polymerization assay kit, by Cytoskeleton (1 mentions)
2 7 dichlorofluorescein diacetate, by Merck Group (1 mentions)
Centrifuge 5415r, by Eppendorf (1 mentions)
Oligonucleotide, by Thermo Fisher Scientific (1 mentions)
Icycler, by Bio-Rad (1 mentions)
Kod plus mutagenesis kit, by Toyobo (1 mentions)
Purelink quick plasmid miniprep kit, by Thermo Fisher Scientific (1 mentions)
Dpni, by Thermo Fisher Scientific (1 mentions)
Pfu turbo dna polymerase, by Agilent Technologies (1 mentions)
3130 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
25 protocols using spectrafluor plus microplate reader
1
BrdU Cell Proliferation Assay Protocol
2
Quantifying Cell Viability using MTT Assay
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Cell viability was measured with the colorimetric MTT assay (Sigma). We seeded MCPIP1, D141N and PURO cells (5 × 103 per well) in 96-well plates and stimulated them with doxycycline (DOX) for 24 h. Then, MTT assay was performed at 24, 48 and 72 h and measured the absorbance at 570 nm relative to 690 nm in a Tecan Spectra Fluor Plus Microplate Reader (Tecan Group Ltd.). The experiment was performed four times with quintuplicate samples in each experiment. Data was expressed as the mean absorbance of each sample relative to PURO cells.
3
ATPLite Assay for Cell Viability
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Cell viability was analyzed with ATPLite—Luminescence ATP Detection Assay System (PerkinElmer). Cells were plated on 96-well white plates (5 × 103 per well), and after 24 h, MCPIP1 expression was stimulated with doxycycline. Quantification of intracellular ATP content was carried out according to the manufacturer’s instructions after 48 h of exposure. The luminescence was measured using the Tecan Spectra Fluor Plus Microplate Reader (Tecan Group Ltd., Männedorf, Switzerland).
4
Colorimetric MTT Cell Viability Assay
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Cell viability was measured using the colorimetric MTT assay as described [54 (link)]. Absorbance was measured using the Tecan Spectra Fluor Plus Microplate Reader (Tecan Group Ltd.) at 570 nm with the reference wavelength of 500 nm. Three independent experiments were performed, each in quintuplicate.
5
BrdU Cell Proliferation Assay
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Cell proliferation was measured with the BrdU Cell Proliferation Assay Kit (Roche). We seeded 5 × 103 MCPIP1, D141N and PURO cells in 96-well plates and stimulated them with 1 µg/ml doxycycline (BioShop) for 24 h. Then, the cells were incubated with 10 μM BrdU for 8 h and the incorporated BrdU was quantified according to the manufacturer’s instructions by measuring the chemiluminescence with the Tecan Spectra Fluor Plus Microplate Reader (Tecan Group Ltd.). The experiment was performed four times and the data was presented as the mean luminescence value (percentage) for each sample relative to the PURO cells. Mean luminescence of the PURO cells was set as 100%.
6
Kinetic Assay for Human Butyrylcholinesterase
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hBChE functional activity was continuously
measured spectrophotometrically (Tecan SpectraFluorPLUS microplate
reader, Tecan Group Ltd.) with an Ellman assay.16 (link) Briefly, BTC (Fluka, St. Louis, MO) was used as a substrate
for WT and hBChE variants over a final concentration range of 0.01–100
mM. Incubations were conducted in PBS at 25 °C in the presence
of 0.4 mM DTNB (Sigma-Aldrich). A highly purified hBChE enzyme (i.e.,
WT, Y332S, D340H, Y332S/D340H, or G117H) (3.5 × 10–3 IU) was added to initiate the incubation, and hydrolysis was
continuously monitored by recording the absorbance at 405 nm. The
functional activity was calculated from the molar extinction coefficient
of 13600 M–1 cm–1.16 (link) Observed rates of hydrolysis were fit to eq1 , which describes
the activation of the enzyme (when b > 1) at relatively
high BTC concentrations from the binding of the substrate to a secondary
binding site27 (link) that afforded best-fit
kinetic values and
relevant standard errors. For the purpose of analysis, b was fixed at 1.7 and Kss was constrained
to be greater than zero.12
measured spectrophotometrically (Tecan SpectraFluorPLUS microplate
reader, Tecan Group Ltd.) with an Ellman assay.16 (link) Briefly, BTC (Fluka, St. Louis, MO) was used as a substrate
for WT and hBChE variants over a final concentration range of 0.01–100
mM. Incubations were conducted in PBS at 25 °C in the presence
of 0.4 mM DTNB (Sigma-Aldrich). A highly purified hBChE enzyme (i.e.,
WT, Y332S, D340H, Y332S/D340H, or G117H) (3.5 × 10–3 IU) was added to initiate the incubation, and hydrolysis was
continuously monitored by recording the absorbance at 405 nm. The
functional activity was calculated from the molar extinction coefficient
of 13600 M–1 cm–1.16 (link) Observed rates of hydrolysis were fit to eq
the activation of the enzyme (when b > 1) at relatively
high BTC concentrations from the binding of the substrate to a secondary
binding site27 (link) that afforded best-fit
kinetic values and
relevant standard errors. For the purpose of analysis, b was fixed at 1.7 and Kss was constrained
to be greater than zero.12
7
MTT Cell Viability Assay
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Five-thousand cells per well were seeded in 96-well plates. Then 0.5 mg/mL of MTT solution (Sigma) was added for 2 hours. Reaction was stopped by adding 5 mmol/L HCl (POCH) in isopropanol (Chempur) and stirred for 30 minutes to dissolve the formazan crystals. Absorbance was measured at 570 nm with the reference wavelength of 500 nm using Tecan Spectra Fluor Plus Microplate Reader (Tecan Group Ltd.). Measurements were performed 24, 48, 72, and 96 hours after seeding. Three independent experiments were performed in triplicate.
8
Engineered E. coli for αKG Uptake
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The E. coli strain JW2571 (Baba et al., 2006 (link)), in which the only endogenous αKG transporter under aerobic conditions KgtP (Seol and Shatkin, 1991 (link)) was knocked out, was transformed with a pGEM-5Zf(+) vector encoding LaINDY. Transformed cells were grown in LB broth with ampicillin to an OD595 ~0.8, then diluted to an OD595 of 0.1. Dilute cells were inoculated into eM9 media supplemented with 50 mM αKG (Rhie et al., 2014 (link)), and cell growth monitored using a Tecan SPECTRAFluor Plus microplate reader (Männedorf, Switzerland) incubated at 37°C.
9
Caspase-3/7 and Cell Viability Assays in Trpv4 KO PTCs
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96-well clear plates were seeded with 1 × 104 WT or Trpv4 KO PTC/well. Cells were allowed to attach overnight. On average, cells were at 50% confluency before addition of 300 μM CoCl2. Twenty-four hours after CoCl2 treatment the cells were assayed by the Caspase-Glo 3/7 or by the CellTiter-Glo Luminescent Cell Viability Kit according to manufacturer’s instructions (Promega). After 10 min on a plate shaker at room temperature, 90% of the lysate volume was transferred to a 96-well solid-white plate (Nunc). Cell lysates were analysed in a SpectraFluor Plus microplate reader (Tecan), and blank values were subtracted from data points. Relative fluorescence intensity of treated cells was normalized by their respective untreated control cells to normalize for possible seeding density differences between WT and Trpv4 KO PTC. In case of the CellTiter-Glo Luminescent Cell Viability Assay an ATP standard curve was measured in every experiment.
10
Quantifying Liver Cytokines in Mice
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The lysates for ELISA were isolated from the right median lobe in 1% Triton in PBS with protease inhibitors, then centrifuged for 10 min, 4 °C, 10,000× g. To evaluate levels of cytokines in mouse livers (TGFβ and IL-1β), DuoSet ELISA assays were performed (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Absorbance was measured at 450 nm; 540 nm was used as reference using a Tecan Spectra Fluor Plus Microplate Reader (Tecan, Männedorf, Switzerland).
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