Cells were treated with glucose deprivation with or without siRNA targeting AMPK or TSC2. Using a cell proliferation kit (Cat # 11465007001, MTT, Sigma-Aldrich), 24 h after the addition of media with or without glucose, 200 μL of MTT solution was added to each well. Cells were incubated for 4 h at 37°C. Two milliliters MTT solubilization buffer were then added to each well, and cells were rocked to ensure adequate distribution. Cells were incubated overnight at 37°C. The cell media with buffer was thoroughly mixed to assure solubilization of the colorimetric marker, and absorbance was read at 595 nm. A negative control, with cell media and buffer only, no cells, was read alongside for normalization.
Cell proliferation kit
Manufactured by Merck Group
Sourced in United States, Germany
The Cell Proliferation Kit is a laboratory equipment product designed to measure cell proliferation. It provides a quantitative assessment of cell growth and viability.
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Lab products found in correlation
Live dead bacterial viability kit, by Thermo Fisher Scientific (2 mentions)
Microplate reader, by Agilent Technologies (2 mentions)
Synergy h1 microplate reader, by Agilent Technologies (1 mentions)
Md spectramax m5, by Molecular Devices (1 mentions)
Ly294002, by Cell Signaling Technology (1 mentions)
96 well culture plate, by Thermo Fisher Scientific (1 mentions)
Milli q deionized water, by Merck Group (1 mentions)
Lauric acid, by Merck Group (1 mentions)
Vascular endothelial growth factor (vegf), by R&D Systems (1 mentions)
Varioskan lux microplate reader, by Thermo Fisher Scientific (1 mentions)
Camptothecin, by Merck Group (1 mentions)
Gsh glo glutathione assay, by Promega (1 mentions)
Inverted microscope, by Zeiss (1 mentions)
Spark multimode microplate reader, by Tecan (1 mentions)
Confocal microscopy, by Zeiss (1 mentions)
18 protocols using cell proliferation kit
1
MTT Assay for Glucose Deprivation
2
Evaluating Macrophage Viability with H. polygyrus Antigens
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The effect of H. polygyrus antigens on macrophage viability was assessed using the MTT assay (Cell Proliferation Kit, Sigma-Aldrich). MTT is chemically 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. First, 1 × 105 cells/well were seeded in 96-well adhesion plates and 200 µL of DMEM medium supplemented with medium conditioned with L929 cells (20%), BCS (10%), and penicillin and streptomycin (1%) was added to each well. The final concentration of two types of somatic antigen solutions (differing in LPS concentration) and secretory-excretory antigens was 10 µg/mL. In addition to the negative control without antigens, a control with LPS (0.1 EU/mL) was performed, which was the highest LPS concentration measured among the tested antigens. The cells were incubated for 24 and 48 h at 37 °C and 5% CO2. The viability of macrophages was assessed by measuring the absorbance at 570 nm using the Infinite Plate Reader 2000 (Tecan, Männedorf, Switzerland). The experiment was performed 3 times with 4 technical replicates (n = 12).
3
Macrophage Viability Assay with GM Treatments
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The viability of M0 macrophages treated with varying concentrations of GMs or their extracts for 72 h was determined using the Cell Proliferation kit (XTT assay, Sigma-Aldrich, St. Louis, Missouri, USA) Briefly, after each incubation period (24, 48, and 72 h), XTT was added to the respective wells and the cells were incubated for a minimum of 4 h at 37 °C as per the manufacturer's instructions. The absorbance of the reduced formazan product formed was then measured at wavelengths of 450 nm and 630 nm using a microplate reader (Synergy H1 microplate reader, Biotek Instruments, USA) and the % viability was calculated using the following equation:
4
High Glucose-Induced Osteoblast MTT Assay
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The osteoblasts were cultured in 96-well plates (at 103 cells/well). An MTT assay was conducted after 1, 4 or 7 days of exposure to high glucose (16.5 mM) using a cell proliferation kit (Sigma, Oakville, ON, Canada) according to the manufacturer's instructions. After 24 h, the culture medium was replaced with 100 ml of MTT (0.5 mg/ml). The black crystals that formed after 2–3 h were dissolved with acidified isopropanol and the absorbance was measured at 570 nm using a microplate reader (MD SpectraMax M5; Molecular Devices, Sunnyvale, CA, USA).
5
MTT Assay for HCC Cell Proliferation
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The proliferation of HCC cells was measured in sextuplicate by MTT assay by using Cell Proliferation Kit (Sigma-Aldrich) according to the manufacturer's instructions.
6
Rg1 Purity Analysis by HPLC
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Rg1 (purity > 98%) with high‐performance liquid chromatographic analysis was obtained from the Jilin University School of Pharmaceutical Sciences. Ly294002 was obtained from Cell Signaling Technology. The Cell Proliferation Kit (MTT) was obtained from Sigma.
7
Cell Proliferation Assay using MTT
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Reduction of MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium1 bromide) to its formazan salt was used as an estimation cell proliferation (Cell Proliferation Kit; Sigma-Aldrich). Briefly, 4000 cells/well were seeded in 96 well plates. At the time of analysis, cells were incubated with 1 mg/mL MTT for 2.5 h. The reaction was stopped by incubation in 100 µL DMSO for 20 min. Absorbance at 570 nm was taken as an indirect estimation of the proliferation rate of viable cells.
8
Selective Elimination of Der p 1-Reactive Cells
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The therapeutic potential of proDer p 1-ETAʹ and proDer p 1-SNAP-AURIF for the selective elimination of Der p 1 reactive hybridoma cells was compared by a cell viability assay (XTT assay) using the cell proliferation kit (Sigma-Aldrich, USA). Briefly, 5 × 103 cells were seeded/well overnight in 96-well plates in complete medium (DMEM medium supplemented with 10% (v/v) FBS and 1% penicillin streptomycin) at 37°C and 5% CO2. After 24 h, the cells were treated with decreasing concentration of the allergen-rIT or allergen-drug conjugate for 48 h after which the XTT reagent was added to each well and incubated for another 4 h. Zeocin® (100 μg/ml) was used as a control to achieve 100% cell killing. The readout was conducted by measuring absorbance of the reduced XTT reagent at 450 nm with a reference of 655 nm on a spectrophotometer (Bio-Rad).
9
Evaluating Cholangiocarcinoma Cell Viability
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A human cholangiocarcinoma cell line 1 was incubated in a 96-well culture plate (Nunc, Rochester, New York, USA) with 1 × 104 cells per well for 24 h at 37°C with 5% CO2. The films of the same size were manufactured with different amounts of SHEC and PTX, as previously described. They were subsequently dissolved in dimethyl sulfoxide and were inserted into the wells of the plate. After 24 h of incubation, the numbers of viable cells were calculated using a Cell Proliferation Kit (MTT; Sigma-Aldrich, St. Louis, MO, USA) and measured at a wavelength of 570 nm.
10
Peptide Amphiphile Synthesis and Characterization
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Wang resin, 9-Fluorenylmethoxycarbonyl (Fmoc) and tert-butoxycarbonyl (Boc)-protected amino acids, and 2-(1H-benzotriazol-1-yl)-1,1,3,3 tetramethyluronium hexaf luoro-phosphate (HBTU) for peptide amphiphile synthesis were purchased from NovaBiochem (Alexandria, NSW, Australia). Lauric acid and N,N- diisopropylethylamine (DIEA) were purchased from Merck (Rahway, NJ, USA). Other chemicals were purchased from Alfa Aesar (Haverhill, MA, USA) or Sigma-Aldrich (St. Louis, MI, USA) and used without any purification. We ensured that all chemicals were of analytical grade and were used without further purification.
Millipore Milli-Q deionized water were used during the experiments with a resistance of 18 MΩ·cm. Vascular endothelial growth factor (VEGF) was purchased from R&D Systems (293-VE-050/CF). The cell proliferation kit was purchased from Sigma (Cat. No. 11 465 007 001), and the goat-anti-human IgG/F (ab)2 antibody labeled with horseradish peroxidase was purchased from Thermo Fisher (Catalog #31122), Waltham, MA, USA.
All cell culture chemicals were purchased from Biological Industries (Kibbutz Beit Haemek, Israel), and cell culture consumables were purchased from Nest Scientific USA Inc. (Woodbridg, NJ, USA). Finally, the Lucentis used in our study was donated by Gazi University, Department of Ophthalmology.
Millipore Milli-Q deionized water were used during the experiments with a resistance of 18 MΩ·cm. Vascular endothelial growth factor (VEGF) was purchased from R&D Systems (293-VE-050/CF). The cell proliferation kit was purchased from Sigma (Cat. No. 11 465 007 001), and the goat-anti-human IgG/F (ab)2 antibody labeled with horseradish peroxidase was purchased from Thermo Fisher (Catalog #31122), Waltham, MA, USA.
All cell culture chemicals were purchased from Biological Industries (Kibbutz Beit Haemek, Israel), and cell culture consumables were purchased from Nest Scientific USA Inc. (Woodbridg, NJ, USA). Finally, the Lucentis used in our study was donated by Gazi University, Department of Ophthalmology.
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