The WST-1 metabolic assay was performed as previously published (36). Cells were exposed to IL-4 + IL-13, IL-22, or IFNγ at doses ranging from 3.1 to 200 ng/mL, or JAKi at 400 nM, and viability was assessed 24 or 48 h later. Absorbance readings (450 nm and 620 nm) were taken using a Spectramax i3x Multi-Mode Plate Reader (Molecular Devices, San Jose, CA, USA). Values were normalized to the percent of media control. LDH assays were performed by plating N/TERT-2G cells at a density of 75,000 cells per well in a 96-well plate. These cells were grown to confluency and then exposed to cytokines or JAKi treatments as noted above. Supernatants were collected from each well and the LDH assay was performed (Cytotoxicity Detection KitPLUS LDH, Millipore Sigma Saint Louis, MO, USA). The LDH detection reaction was conducted at room temperature and terminated by the addition of Stop Solution after 10 min. Readings (absorbance at 490 nm and 620 nm) were taken using a Spectramax i3x Multi-Mode Plate Reader (Molecular Devices, San Jose, CA, USA). The absorbance values from media-only wells were subtracted from each condition, and all values were normalized to a lysed control sample (representing 100% death) generated through addition of a 1:10 dilution of the provided lysis buffer.
Spectramax i3x multi mode plate reader
Manufactured by Molecular Devices
Sourced in United States
The SpectraMax i3x Multi-Mode Plate Reader is a versatile instrument designed for various laboratory applications. It is capable of performing absorbance, fluorescence, and luminescence measurements in microplate format. The SpectraMax i3x provides a range of detection modes to support diverse experimental needs.
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Lab products found in correlation
Spectramax 190 plate reader, by Molecular Devices (2 mentions)
Realtime glo annexin 5 apoptosis assay, by Promega (2 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (2 mentions)
Toxilight non destructive cytotoxicity bioassay, by Lonza (2 mentions)
Raltegravir, by Santa Cruz Biotechnology (2 mentions)
Passive lysis buffer (plb), by Promega (2 mentions)
Dc protein assay, by Bio-Rad (2 mentions)
Luciferase assay system, by Promega (2 mentions)
Celltitre blue cell viability assay, by Promega (1 mentions)
Cytotoxicity detection kitplus ldh, by Merck Group (1 mentions)
Enzchek pyrophosphate assay kit, by Thermo Fisher Scientific (1 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
Celltiter glo, by Promega (1 mentions)
Voriconazole, by Merck Group (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Vinculin, by Cell Signaling Technology (1 mentions)
Image studio lite software, by LI COR (1 mentions)
Mops buffer, by Thermo Fisher Scientific (1 mentions)
Nestin, by Cell Signaling Technology (1 mentions)
Scanlater, by Molecular Devices (1 mentions)
23 protocols using spectramax i3x multi mode plate reader
1
Cytokine-Induced Cell Viability Assay
2
Evaluating Cell Viability Responses
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The effect of the released PTN on cells viability have been analyzed using an indirect viability assay performed on both HUVECs and HUASMCs. Briefly, cells have been seeded at a concentration of 20,000 cells/cm2 in 96 well culture plates. After 24 h of incubation with HUVEC-M199 or HUASMC-M199, depending on the cell type used, at 37°C in a saturated atmosphere at 5% CO2 to allow the adhesion of the cells, media has been removed and cells have been incubated for 24 h in presence of the different conditioned media collected as previously described. Cells cultivated in P/S-M199 medium have been used as a positive control (CTRL Cell). After the treatment, the conditioned media have been removed and cells have been incubated with a resazurin solution for 4 h. After the incubation, the highly fluorescent resorufin product obtained by the reduction of the resazurin was collected and fluorescence intensity at a 545 nmex/590 nmem wavelength was measured with a SpectraMax i3x Multi-Mode Plate Reader (Molecular Devices, San Jose, California, USA). Fluorescence intensity is proportional to cell viability. Data has been normalized toward the CTRL Cell condition.
3
Cell Proliferation and Apoptosis Assays
4
Extraction and Quantification of Muscle Proteins
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Sarcoplasmic (water-soluble) proteins and myofibrillar (salt-soluble) proteins were extracted from rainbow trout raw material as described by Anderson and Ravesi [15 ] and Licciardello et al. [16 ]. About two grams of minced muscle was homogenized in 40 ml of buffer (50 mM KH2PO4, pH7), and centrifuged at 4 °C for 20 min at 4100 g. The supernatant containing the sarcoplasmic proteins was decanted through glass wool and the volume was made up to 50 ml with buffer. To collect the myofibrillar proteins, the sediment was re-homogenized in 40 ml of buffer (50 mM KH2PO4, 0.6M KCl, pH7), centrifuged, decanted through glass wool, and volume was made up to 50 ml with buffer.
The protein content in the extract was determined in triplicate in suitable dilutions of both fractions by the method of Bradford [17 (link)]. Diluted color reagent (5 ml) was added to blank (distilled H2O), standards and samples, and absorbance at 595 nm was measured after 5 min using SpectraMax i3x Multi-Mode Plate Reader (Molecular Devices, LLC., San Jose, USA).
The protein content in the extract was determined in triplicate in suitable dilutions of both fractions by the method of Bradford [17 (link)]. Diluted color reagent (5 ml) was added to blank (distilled H2O), standards and samples, and absorbance at 595 nm was measured after 5 min using SpectraMax i3x Multi-Mode Plate Reader (Molecular Devices, LLC., San Jose, USA).
5
Quantifying Phenolic Content in Protein Extracts
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Deionized water, Folin-Ciocalteu reagent and the sample (protein extract from raw material and FPH) was mixed by vortexing. After 3 min, 1 ml of 20% Na2CO3 was added, and the volume was filled up to 10 ml by adding H2O. A calibration with concentrations ranging from 0.5 mM to 2 mM propyl gallate was prepared using the same reagents and procedure as water. The absorbance at 725 nm was read on SpectraMax i3x Multi-Mode Plate Reader (Molecular Devices, LLC., San Jose, USA) after 1 h of incubation at room temperature. For calculation, formula (2) was used. where C = total phenolic content in mmol/g, C1 = concentration of propyl gallate established from the calibration curve, V = volume of extract in ml, and m = the weight of the sample in g.
6
Malate Dehydrogenase-Coupled Enzyme Assay
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Malate dehydrogenase-coupled enzyme assays were performed at 22 °C in a 96-well plate format for a total reaction volume of 200 μL. Assay conditions consisted of 100 mM Tris (pH 7.8), 7 mM MgCl2, 150 mM KCl, and 0.5% Triton x-100. First, 20 μL of compound was added to obtain the desired final concentration; 20 μL of malate dehydrogenase (MDH) was added such that the final concentration in the assay was 20 U/mL; and lastly, 140 μL of substrates was added to initiate the reaction (HCO3−, ATP, and NADH to a final assay concentration of 15 mM, 2.5 mM, and 0.25 mM, respectively). To account for the possible compound inhibition of MDH, the procedure was modified such that 20 μL oxaloacetate was added to a final concentration of 30 mM, in place of PC. Reagents were dispensed manually by a hand-held, multi-channel micropipette, and absorbance measurements were recorded at 340 nM with a Molecular Devices SpectraMax i3x Multi-mode plate reader.
7
Cell Proliferation and Apoptosis Assay
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Cells proliferation was determined using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega) according to the manufacturer's instructions. Briefly, the cells were incubated with tetrazolium compound [3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium for 1 hour and the quantity of the formazan product was measured as the absorbance at 490 nm on a SpectraMax 190 plate reader (Molecular Devices). Apoptosis was assessed using the RealTime‐Glo Annexin V Apoptosis Assay (Promega) according to the manufacturer's instructions. Luminescence over 30 minutes was read on a SpectraMax i3x Multimode plate reader (Molecular Devices). Data are reported at the maximal velocity (Vmax).
8
Cell Viability Assay Protocol
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Cell viability was determined using the CellTitre-Blue Cell Viability
Assay from Promega as per manufacturer's protocol. Briefly, 10,000
cells were seeded in 96 well plate and different dilutions of the
drugs were added. Plates were incubated for 48h at 37°C. 20µL of
the CellTitre-Blue reagent was added and incubated for additional
2h. Fluorescence was measured at 540Ex/590Em on SpectraMax i3x
Multi-Mode plate reader (Molecular Devices, USA). The mean
inhibitory concentration of the drug (IC50) was obtained using the
non-linear regression model.
Assay from Promega as per manufacturer's protocol. Briefly, 10,000
cells were seeded in 96 well plate and different dilutions of the
drugs were added. Plates were incubated for 48h at 37°C. 20µL of
the CellTitre-Blue reagent was added and incubated for additional
2h. Fluorescence was measured at 540Ex/590Em on SpectraMax i3x
Multi-Mode plate reader (Molecular Devices, USA). The mean
inhibitory concentration of the drug (IC50) was obtained using the
non-linear regression model.
9
High-throughput Antifungal Compound Screening
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The NCI small molecule screen was performed by dispensing 80 μl of screening medium into plates containing compound, then dispensing 20 μl of conidial suspension (0.01% Tween 80 with 5 × 105 conidia/ml) for final concentrations of 20 μM compound, 2% DMSO, and 1 × 105 conidia/ml. Cayman kinase inhibitor and FDA-approved libraries were screened at 25 μM compound, 1% DMSO and 1 × 105 conidia/ml concentrations. Plates were incubated at 37°C for 16 h, then allowed to come to room temperature for 1.5 h before addition of 100 μl AK detection reagent per well (Toxilight non-destructive cytotoxicity bioassay, Lonza, LT07). Plates were incubated for 1 h at room temperature, then luminescence was measured with 140 ms integration on a SpectraMax i3X Multi-Mode plate reader (Molecular Devices). Data were log transformed and ZR scores for each well were calculated as previously described (22 (link)). Hits were validated by testing compounds under the same screening conditions, with DMSO controls filling the remainder of the plates. For Cayman libraries, hits were also validated by growing wells without compound using the above conditions, then adding compound just before addition of AK detection reagent. Data were log transformed and ZR scores were calculated for each well (22 (link)).
10
Kinetic Characterization of Kbc1 Acetoacetate Utilization
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Kbc1 activity was detected using our assay previously described for Acs1 activity except sodium acetoacetate was used in place of sodium acetate (18 (link)). Michaelis-Menten constants were determined for the acetoacetate, CoA, and ATP substrates by first determining the optimal concentrations of these substrates so that they would be in excess without, in the case of CoA, being high enough to inhibit the reaction. Substrates provided in excess allow the apparent Km to closely approximate the actual Km. The EnzChek Pyrophosphate assay kit (Thermo) was used with reagents prepared by manufacturer’s standards, and with 4 mM MgCl2, 10 mM DTT, 4.5 μg/mL Kbc1, 100 μM CoA, and 200 μM ATP per 50 μL reaction. The reagents were mixed and aliquoted at room temperature, allowed to incubate for 15 min at 37°C to mop background phosphate contamination, then acetoacetate (final concentration of 1 mM) was added to the reaction, and the plate was read continuously for 40 min at 37°C in a SpectraMax i3X Multi-Mode plate reader (Molecular Devices) at absorbance 360 nm. To test possible substrates, a dilution series of propionate, butyrate, 3-hydroxybutyrate, or acetate was added in place of acetoacetate.
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