To confirm osteogenic differentiation and to determine the level of activity of the differentiated hMSCs, two assays were performed: alkaline phosphatase (ALP) activity and total protein content (micro-BCA assay). Alkaline phosphatase activity was assessed using the Alkaline Phosphatase Colorimetric Assay Kit (Abcam ab83369). According to standard protocols, after the cell's exposure to osteogenic differentiation medium for periods of 3 days, cells were washed twice with PBS. Then, 50 μL of the cell lysate with assay buffer was added to a 96 well-plate and 50 μL p-nitrophenyl phosphate (pNPP). The samples were incubated at 25 °C for 60 min and protected from light. In the last step, 20 μL stop solution was added to the wells, then; the plate was read at 405 nm in a microplate reader (Synergy LX Multi-Mode Reader from BioTek® (Model SLXFA). Alkaline phosphatase (ALP) activity was normalized by total protein content (micro-BCA assay). The total protein content was determined according to the protocol of the manufacture 150 μL of sample was placed in a 96 well-plate with 150 μL of working reagent made from a micro-BCA protein assay kit (Thermo Scientific). The well plate was covered with foil and incubated at 37 °C for 2 h. Absorbance was read at 562 nm using a BioTek Multi-Mode Microplate Reader (Model Synergy™ 2).
Model synergy 2
Manufactured by Agilent Technologies
Sourced in United States
The Synergy 2 is a multi-mode microplate reader from Agilent Technologies. It is designed to measure various types of biological and chemical assays in a 96-well or 384-well microplate format. The Synergy 2 supports absorbance, fluorescence, and luminescence detection modes.
Automatically generated - may contain errors
Lab products found in correlation
Alkaline phosphatase assay kit colorimetric, by Abcam (1 mentions)
Synergy lx multi mode reader, by Agilent Technologies (1 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Nonesterified fatty acids, by Abcam (1 mentions)
Insulin, by ALPCO (1 mentions)
Prestoblue cell viability assay, by Thermo Fisher Scientific (1 mentions)
Bms607 3, by Thermo Fisher Scientific (1 mentions)
Bms6002, by Thermo Fisher Scientific (1 mentions)
Cck 8 reagent, by Dojindo Laboratories (1 mentions)
Celltiter 96 aqueous one solution reagent, by Promega (1 mentions)
8 protocols using model synergy 2
1
Osteogenic Differentiation Assays for hMSCs
2
Comprehensive metabolic profiling in rats
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After an overnight fast, rats were briefly gassed with CO2 and euthanized by cervical decapitation. Mixed blood was collected in chilled tubes containing EDTA and centrifuged at 12,000 rpm for 4 min. The plasma was removed and stored at −80 °C for measurement of glucose (Abcam, Cambridge, MA, USA), insulin (Alpco Diagnostics, Salem, NH, USA), non-esterified fatty acids (Abcam, Cambridge, MA, USA), and FC (Abcam, Cambridge, MA, USA) following the manufacturer’s guidelines and using a multi-mode microplate reader (Biotek Instruments Inc., model Synergy 2, Winooski, VT, USA). Livers and kidneys were excised, weighed, and snap frozen in liquid nitrogen. Inguinal, peritoneal, and visceral fat pads were carefully excised and weighed to determine total fat pad weight.
3
PrestoBlue Viability Assay for hMSCs
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For hMSCs viability, the PrestoBlue™ cell viability assay from Invitrogen (Cat. #A13261) was used. hMSCs (10000 cells/cm2) were seeded on each surface prepared on a 96 well-plate, and cell viability was measured after 3 days of culture as described in our previous works [29 (link),40 (link)]. Briefly, the cell culture medium was removed after 3 days, and 100 μL per well containing 90% fresh cell medium and 10% PrestoBlue reagent were added. The plate was incubated for 3 h, and the fluorescence intensity measurement was determined using a BioTek Multi-Mode Microplate Reader (Model Synergy™ 2) with excitation/emission of 560/590 nm.
4
Hippocampal Oxidative Stress Assay
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The hippocampus was homogenated in 0.1 M phosphate buffer (pH 7.4) and ~0.25 mg/mL was added to 0.2 mL of the same buffer containing 10 mM glutamate, 10 mM pyruvate, 4 mM malate, and 1 U/mL peroxidase at 37 °C. The reaction trigger was initiated by the addition of 2 µM Amplex Red, and hydrogen peroxide (H2O2) production was monitored by spectrofluorimetry at 563/587 nm (ex/em) [34 (link)] in a Model Synergy 2 fluorescence spectrophotometer (Biotek, Winooski, VT, USA) with continuous stirring. The results were analyzed through the slope variation rate, calculated by the difference between final fluorescence and initial fluorescence (unit of fluorescence—UF) divided by time and normalized by protein concentration analyzed by the Bradford protocol [31 (link)].
5
Cytokine Quantification in Cell Lines
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Following the manufacturer's instructions, the levels of TNF-α, IL-1β, or IL-18 in BV2 or HT-22 cell culture supernatants were determined by ELISA (Cat. no. BMS607-3 and BMS6002, eBioscience, San Diego, CA, USA, for TNF-α and IL-1β, and Cat. no. YJ003057, Mlbio, Shanghai, China, for IL-18). The absorbance was measured using a microplate reader at 450 nm (Model Synergy 2, BioTek, YT, USA).
6
Cell Membrane Integrity Evaluation
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LDH is an enzyme that is normally found in the cytoplasm and is released into the medium when the cell membrane is disrupted, thus, the quantification of LDH release can be used to evaluate the integrity of cell membranes. After LPS and ATP treatment, using an LDH Assay kit (Cat. no. K726, BioVision, Mountain View, CA, USA) in accordance with the manufacturer's instructions, the cell supernatants were gathered for LDH measurement. LDH oxidizes lactate to produce NADH, reacting with WST, which can be measured at 450 nm optical density using an absorbance plate reader. Therefore, the volume of LDH that cells secreted was measured at 450 nm optical density with an absorbance plate reader (Model Synergy 2, BioTek, VT, USA), and then, the values were interpolated using the calibration curve and the LDH release finally was calculated and expressed as the value of U/L, which represents the degree of cell necrosis.
7
Cytotoxicity Assay for BV2 and HT-22 Cells
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To detect the toxicity of emodin or LPS and ATP costimulation, 5 × 10 3 cells per well of plated BV2 or HT-22 cells were cultured in 96-well plates with 100 µL medium overnight. Then, the culture had successive emodin treatments or MCC950, LPS, and ATP at indicated concentrations. 10 µL of the CCK-8 reagent (Cat. no. CK04, Dojindo, Kumamoto, Japan) was added into each well after 24 h, and they were left to incubate for 2 h in dark. A microplate reader (Model Synergy 2, BioTek, Winooski, VT, USA) was used to measure the absorbance at 450 nm.
8
Cytotoxicity Evaluation of Drug-Loaded Membranes
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Both the anti-tumor effects on HCT116 cells and cytotoxicity on NCM460 cells of drug-loaded membranes were determined by MTS assay. Briefly, 5.0×10 3 cells in 100 μL medium were pre--8 -cultivated in each well of the 96-well plates for 12 hrs and then the culture medium was replaced with treatment medium containing experimental samples as described above. After 12, 24, 48, 72, 96 and 120 hrs incubation, the medium was aspirated and cells were washed with PBS for three times.
Subsequently, 100 μL fresh serum-free medium supplemented with 20 μL CellTiter 96® AQueous One Solution Reagent (Promega) was added and incubated for another 3hrs. The absorbance values were measured at 490 nm using a microplate reader (Model Synergy 2, Bio-Tek, Vermont, USA) in a dark environment. Cell viability was calculated using Eq. ( 1) below:
(
Where the test groups were the cells subjected to different treatments, and the control groups were the untreated cells.
Subsequently, 100 μL fresh serum-free medium supplemented with 20 μL CellTiter 96® AQueous One Solution Reagent (Promega) was added and incubated for another 3hrs. The absorbance values were measured at 490 nm using a microplate reader (Model Synergy 2, Bio-Tek, Vermont, USA) in a dark environment. Cell viability was calculated using Eq. ( 1) below:
(
Where the test groups were the cells subjected to different treatments, and the control groups were the untreated cells.
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