Cell metabolism was studied after labeling hMSCs with SiC nanomaterials using CellTiter 96® AQueous One Solution cell proliferation assay (MTS, Promega). After co-incubation with SiC nanomaterials for 4 hours, 20 μL of the assay reagent was pipetted into the samples in 100 μL of growth media. This was allowed to incubate under standard conditions for 4 hours. We then transferred 80 μL of the sample solutions to a new plate and read the absorbance at 490 nm on a spectrophotometer (SpectraMax M5, Molecular Devices). Cell viability was studied with the calcein AM cell viability assay kit (Biotium). Labeled cells were incubated with 100 μL of the reagent assay (containing 2 μM calcein AM) for approximately 1 hour and then the fluorescence was read at 517 nm with an excitation at 494 nm on a spectrophotometer (SpectraMax M5, Molecular Devices). Quantitative ROS measurement was performed by staining cells with 2’,7’-dichlorofluorescin diacetate (DCFDA, Sigma Aldrich) assay. H2O2-treated cells and nontreated cells were included as positive and negative controls, respectively. The fluorescence intensity was read at 530 nm with an excitation at 485 nm on a spectrophotometer (SpectraMax M5, Molecular Devices).
Spectramax m5
Manufactured by Molecular Devices
Sourced in United States, China, Japan, Switzerland, United Kingdom, Canada, Germany, Brazil, Uruguay
The SpectraMax M5 is a multi-mode microplate reader designed for absorbance, fluorescence, and luminescence measurements. It provides a diverse range of capabilities to support various applications in life science research and drug discovery.
Automatically generated - may contain errors
Lab products found in correlation
Methyl thiazolyl tetrazolium (mtt), by Merck Group (36 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (33 mentions)
96 well plate, by Corning (20 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (19 mentions)
Dual luciferase reporter assay system, by Promega (18 mentions)
Cell counting kit 8 (cck8), by Beyotime (17 mentions)
Cck 8, by Dojindo Laboratories (17 mentions)
Prism 5, by GraphPad (15 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (15 mentions)
Celltiter glo, by Promega (13 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (13 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (13 mentions)
Softmax pro, by Molecular Devices (12 mentions)
Prism 6, by GraphPad (12 mentions)
Cck 8, by Beyotime (11 mentions)
Fitc dextran, by Merck Group (11 mentions)
Prism 7, by GraphPad (11 mentions)
Alamar blue, by Thermo Fisher Scientific (10 mentions)
Prism 8, by GraphPad (10 mentions)
Celltiter blue cell viability assay, by Promega (9 mentions)
2 878 protocols using spectramax m5
1
Evaluating SiC Nanomaterial Impact on hMSCs
2
Caspase and PARP Activity Assays
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The activities of caspase-9 and caspase-3 were determined using the caspase colorimetric test kits based on spectrophotometric detection of the chromophore p-nitroanilide (p-NA) as a result of the cleavage of the labeled substrate acetyl-Leu-Glu-His-Asp-pNA and Asp-Glu-Val-Asp-pNA, respectively. The assay kits for caspases-9 (Cat. # ab65608) and caspase-3 (Cat. # ab39401) were purchased from Abcam plc. (Cambridge, MA, USA). Free pNA was identified with a microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA, USA) at 405 nm.
The PARP/Apoptosis colorimetric assay kit (R&D Systems, Minneapolis, MN, USA; Cat. # 4684-096-K) was used for the determination of PARP activity based on a semi-quantitative measurement of the amount of poly (ADP-ribose) deposited on the immobilized histone proteins. The absorbance values at 450 nm were obtained from a microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA, USA). All values were compared to the controls treated by the vehicle.
The PARP/Apoptosis colorimetric assay kit (R&D Systems, Minneapolis, MN, USA; Cat. # 4684-096-K) was used for the determination of PARP activity based on a semi-quantitative measurement of the amount of poly (ADP-ribose) deposited on the immobilized histone proteins. The absorbance values at 450 nm were obtained from a microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA, USA). All values were compared to the controls treated by the vehicle.
3
Measuring Cell Proliferation Inhibition
4
Rumen Fluid Analysis of VFAs and Enzymes
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The VFAs concentration of rumen fluids was determined by gas chromatography (GC) with a capillary column (AT-FFAP: 30 m × 0.32 mm × 0.5 μm) using a Shimadzu 2010 plus system (Shimadzu Corporation, Kyoto, Japan) following Liu et al. (2021b) (link). Ammonia-N concentration was analyzed using a spectrometer (SpectraMax M5, Molecular Devices, San Jose, United States) at an absorbance of 630 nm, following Hristov et al. (2001) (link).
Ruminal enzyme activities of carboxymethylcellulase (CMCase), amylase, xylanase, and pectinase were measured by commercial enzyme-linked immune sorbent assay (ELISA) kits (product No: BYE98162, BYE98329, BYE98332, and BYE99021, respectively; Shanghai Bangyi Biological Technology Co. Ltd., Shanghai, China). The rumen fluid was centrifuged at 800 × g for 5 min at 4°C, and the supernatant was shaken ultrasonically for 3 min. The enzyme activities were determined using a spectrometer (SpectraMax M5, Molecular Devices, San Jose, United States) at an absorbance of 450 nm.
Ruminal enzyme activities of carboxymethylcellulase (CMCase), amylase, xylanase, and pectinase were measured by commercial enzyme-linked immune sorbent assay (ELISA) kits (product No: BYE98162, BYE98329, BYE98332, and BYE99021, respectively; Shanghai Bangyi Biological Technology Co. Ltd., Shanghai, China). The rumen fluid was centrifuged at 800 × g for 5 min at 4°C, and the supernatant was shaken ultrasonically for 3 min. The enzyme activities were determined using a spectrometer (SpectraMax M5, Molecular Devices, San Jose, United States) at an absorbance of 450 nm.
5
Membrane Integrity Monitoring in E. coli
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The integrity of the bacterial membrane was monitored by measuring the levels of ATP, DNA and RNA. E. coli was sedimented at 415 × g for 3 min under 25°C and washed two times with distilled water, and then resuspended into 3.6 ml distilled water (1×106 CFU/ml) prior to transfer into a 24-well plate. Subsequently, SA (200 or 400 µM) was added and cells were incubated at 35°C. At predetermined sampling times (0, 0.5, 1, 1.5 and 2 h), the bacterial suspension was centrifuged at 415 g and 25°C for 3 min. A total of 50 µl supernatant was mixed with 100 µl ATP detection buffer (ATP Assay Kit; Beyotime Institute of Biotechnology). The luminous intensity was detected within 10 min by a microplate reader (Spectramax M5; Molecular Devices, LLC). The concentrations of DNA and RNA were measured in another experimental system. Briefly, following two washes with PBS, E. coli cells (1×106 CFU/ml) were plated into a 24-well plate and cultured with SA at 35°C. At various time intervals (0, 2, 4, 6 and 8 h), 100 µl bacterial suspension was collected and centrifuged at 415 g and 25°C for 3 min. The absorbance was determined at 260 nm using a microplate reader (Spectramax M5; Molecular Devices, LLC).
6
Spectrophotometric Assessment of Oxidative Stress
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Oxidative stress-induced cell death was assessed spectrophotometrically via an LDH release assay of cell culture supernatants according to the manufacturer’s instructions. The absorbance of all samples was read at 490 nm using a SpectraMax M5 microplate reader (SpectraMax M5, Molecular Device, USA) within 3 min.
7
Serum Cytokine and Metabolite Profiling
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The concentration of cytokines (including interleukin-1β [IL-1β ], IL-6 , tumor necrosis factor-α [TNF-α ], and IL-10 ) in serum were tested by enzyme-linked immunosorbent assay (ELISA ) kits (Boster, Wuhan, China) and measured by multimode microplate readers (SpectraMax M5, Molecular Devices, San Jose, CA). D-lactic acid (D-LA ) and diamine oxidase (DAO ) in serum were determined by ELISA kits (Enzyme-linked Biotechnology Company, Shanghai, China) and measured by multimode microplate readers (SpectraMax M5, Molecular Devices, San Jose, CA).
8
Urea and Albumin Production in RWV
9
Oxidative Stress Biomarkers Quantification
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GSH, GSSG (oxidized glutathione), and SOD (superoxide dismutase), and reduced and oxidized glutathione were determined in plasma according to Rahman et al. [23 (link)]. The assay is based on the reaction of GSH with 5,5′-dithio-bis (2-nitrobenzoic acid) (DTNB) which produces 5′-thio-2-nitrobenzoic acid (TNB), which has a maximum absorbance at 412 nm and oxidized glutathione-TNB adduct (GS-TNB).
Plasma samples (100 μL) were placed in 5% metaphosphoric acid, centrifuged at 3000 g at 4°C for 10 min, and the upper clear layer was maintained at 0–4°C during the assay. 20 μL of the blank or standard sample was inserted into the reaction plate. The absorbance was measured at 412 nm in a microplate reader (SpectraMax M5, Molecular Devices, USA). For the GSSG assay, 2 μL of 2-vinylpyridine was added to the formation of 100 μL of 2-nitro-5-thiobenzoic acid in the plasma, and the reaction was left for 2 hours in the dark. Then, 20 μL was used for the plate reaction as described above. GSSG was calculated by the rate of formation of 2-nitro-5-thiobenzoic acid compared to the standard curve of GSSG. The reduced GSH was calculated as the total values of glutathione—GSSG. For SOD determination, a commercial kit (Sigma-Aldrich, USA cat. No. 19160) was used according to the manufacturer's instructions and read at 450 nm microplate reader (SpectraMax M5, Molecular Devices, USA).
Plasma samples (100 μL) were placed in 5% metaphosphoric acid, centrifuged at 3000 g at 4°C for 10 min, and the upper clear layer was maintained at 0–4°C during the assay. 20 μL of the blank or standard sample was inserted into the reaction plate. The absorbance was measured at 412 nm in a microplate reader (SpectraMax M5, Molecular Devices, USA). For the GSSG assay, 2 μL of 2-vinylpyridine was added to the formation of 100 μL of 2-nitro-5-thiobenzoic acid in the plasma, and the reaction was left for 2 hours in the dark. Then, 20 μL was used for the plate reaction as described above. GSSG was calculated by the rate of formation of 2-nitro-5-thiobenzoic acid compared to the standard curve of GSSG. The reduced GSH was calculated as the total values of glutathione—GSSG. For SOD determination, a commercial kit (Sigma-Aldrich, USA cat. No. 19160) was used according to the manufacturer's instructions and read at 450 nm microplate reader (SpectraMax M5, Molecular Devices, USA).
10
Enzymatic Activity Quantification
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LDH activity levels were measured using LDH activity kits (cat. no. C0017; Beyotime Institute of Biotechnology). A microplate reader (SpectraMax M5; Molecular Devices, LLC, Sunnyvale, CA, USA) was employed and absorbance was measured at 490 nm. Caspase-3/9 activity was measured using Caspase-3/9 activity kits (cat. nos. C1115 and C1157; Beyotime Institute of Biotechnology). A microplate reader (SpectraMax M5; Molecular Devices, LLC, Sunnyvale, CA, USA) was employed and absorbance was measured at 405 nm.
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