Polarized Caco-2 monolayers were incubated with OMVs (30 μg/insert), 0.1% (v/v) Triton X-100 (Sigma-Aldrich) (positive control) or cell culture medium (negative control) for 4 or 24 h. Medium from ACs was then removed and Transwell filters were placed into an empty 24-well plate. Cell viability was determined using the CyQUANT MTT Cell Proliferation Assay Kit (Invitrogen) according to the manufacturer’s instructions. Absorbance was measured at 570 nm with a plate reader. The assay is based on the conversion of the yellow tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by dehydrogenases of living cells into blue formazan, the amount of which is proportional to the number of living cells (Mosmann, 1983 (link)).
Cyquant mtt cell proliferation assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CyQUANT™ MTT Cell Proliferation Assay Kit is a colorimetric assay that measures the metabolic activity of cells. The assay uses the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to quantify the number of viable cells in a sample.
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Lab products found in correlation
Triton x 100, by Merck Group (1 mentions)
Fluostar omega, by BMG Labtech (1 mentions)
Ox ldl, by Merck Group (1 mentions)
Mtt reagent, by Merck Group (1 mentions)
Microplate reader, by Allsheng (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Cyquant ldh cytotoxicity assay kit, by Thermo Fisher Scientific (1 mentions)
Ebioscience annexin 5 fitc apoptosis detection kit, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
Fluo 4 am, by Thermo Fisher Scientific (1 mentions)
Glass bottomed culture dishes for imaging, by MatTek (1 mentions)
Nano f plus, by Tecan (1 mentions)
Elx800 microplate reader, by Agilent Technologies (1 mentions)
8 protocols using cyquant mtt cell proliferation assay kit
1
Evaluating Caco-2 Cell Viability Using MTT Assay
2
Auditory Cell Proliferation and Viability
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The cell proliferation and viability were measured using CyQUANT™ MTT Cell Proliferation Assay Kit (Invitrogen™, Waltham, MA, USA). Auditory cells were cultured in 96-well plates at 5 × 104 cells/well and incubated for 24 h. Following that, cells were treated with CPE and/or H2O2. After a corresponding incubation period, 10 μL of MTT solution was added to wells. At 4 h, 100 µL of the SDS-HCl solution was added to wells for 4 h at 37 °C. The absorbance was measured at 570 nm using a FLUOstar Omega (BMG Labtech, Ortenberg, Germany). The control group (untreated) was considered as 100% viability.
3
Evaluating Cell Proliferation with MTT Assay
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CyQUANT™ MTT Cell Proliferation Assay Kit (V13154) was bought from Invitrogen, USA. The 1 × 103 HUVECs cells were plated into each well of 96‐well plates. Subsequently, cells were incubated with 0, 20, 40, 60, 80 μg/mL ox‐LDL (Sigma‐Aldrich, MO, USA) for 24 h. Then, 5 mg/mL of MTT reagent (Sigma‐Aldrich, MO, USA) was added and incubated for a further 4 h at 37°C. After being dissolved by 0.01 M HCl‐SDS solution, the dissolved formazan was measured at 570 nm using a Thermomax microplate reader (BioTek EL, Vermont, USA).
4
Cytotoxicity Assay of Neutrophils
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The cytotoxicity assay was conducted using the CyQUANT™ MTT Cell Proliferation Assay Kit from Invitrogen (Cat. No. V13154). Neutrophils were seeded at a concentration of 2 × 105 cells per milliliter in conical tubes. The control and antioxidant-supplemented neutrophils (GSH + NAC and ALL) were pre-incubated for 60 min with their respective media (see materials and methods 4.2). After the pre-incubation period, the neutrophils were washed twice with PBS 1× to remove the media. Then, 100 µL of freshly prepared RPMI 1640 media alone was added to each well of a Nunc Flat-bottom microplate from Thermo Fisher (Cat. No. 168055), with a concentration of 2 × 105 cells per well. Subsequently, 10 µL of the CyQUANT™ MTT reagent was added to each well. The negative control of RPMI 1640 + MTT was included, as indicated by the manufacturer’s protocol. The plate was incubated for 3 h at 37 °C. After the incubation period, the plate was centrifuged for 10 min at 4 °C and 2000 rpm. Then, 85 µL of the total volume was carefully removed, and 50 µL of dimethyl sulfoxide (DMSO) was added to each well. The plate was incubated for 15 min, and each well was resuspended prior to reading. Absorbance readings were performed using a microplate reader (ALLSHENG) at 540 nm.
5
Pigskin-based Biomaterial Evaluation
6
Calcium-Mediated ATP Response in Kidney Cells
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Dulbecco's Modified Eagle Medium (DMEM), DMEM calcium-free media, ATP, and Fluo-4 AM were purchased from Gibco/Thermofisher (Waltham, MA, USA). CyQUANT™ MTT Cell Proliferation Assay Kit was ordered from Invitrogen (St. Louis, MO, USA). Glass-bottomed culture dishes for imaging from MatTek (Ashland, MA). Real-time PCR primers were ordered from Gene Link (NY, USA), cDNA synthesis and realtime PCR kits were from Solis BioDyne (Tartu, Estonia). Cell culture and treatment Human proximal kidney (HK-2) cells obtained from Applied Biological Materials Inc, Canada, were maintained in Dulbecco's Modified Eagle Medium (Gibco/ Thermofisher) with high glucose (4.5 g/L) supplemented with 10% FBS, 2 mM L-glutamine, and 1 % penicillin-streptomycin at 37°C with 5% carbon dioxide. The standard DMEM media contains 1.8 mM calcium as reported in the company datasheet. Adjusting the required calcium concentration was done in calcium-free media from the same company by adding CaCl2. Cells were seeded overnight and left to reach ∼ 60-70% confluency before treatments. The next day, media was removed and replaced either by standard media or calcium-free supplemented with CaCl2 to the required concentration, and the cells were grown for an additional 18-20 h before processing. ATP was added to the media to a final concentration of 50 μM.
7
Cytotoxicity Assay for MCF-7 and MDA-231 Cells
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On a 96-well plate, MCF-7 cells were seeded at 7000 cells/well and MDA-231 cells were seeded at 15,000 cells/well. Cells were treated with 8 points of twofold diluted free Dox or Exo-Dox, with the highest concentrations being 1.2 ng/μl and 6 ng/μl for MCF-7 and MDA-231 cells, respectively. A CyQUANT MTT cell proliferation assay kit (Invitrogen, CA, USA) was used according to the manufacturer's instructions. Briefly, 10 μl of 12 mM MTT was added to wells containing fresh medium and incubated for 3 h at 5% CO 2 and 37°C. All but 25 ul of medium was carefully removed and 100 μl DMSO was added and incubated for 10 min at 37°C to dissolve the insoluble formazan. All wells were gently resuspended to mix and the absorbance was read at 492 nm using the Nano F Plus (Tecan, Zürich, Switzerland).
8
CyQUANT™ MTT Cell Proliferation Assay
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Cell proliferation was determined by CyQUANT™ MTT Cell Proliferation Assay Kit (Invitrogen V13154, St. Louis, MO, USA) following the manufacturer's protocol. Briefly, the cells were plated in a 96-well plate at 10,000 cells per well. After overnight attachment, cells were treated with standard media or calcium-free media adjusted with CaCl2, in the presence or absence of ATP, for 18-20 hours. After treatment, the media was removed and replaced with fresh media before adding 10 μl of the MTT drug reconstituted with PBS to each well. Following incubation at 37°C for 4 h, media was removed leaving around 25 µL in the wells, and 100 μl of DMSO was added to each well followed by pipetting up and down to mix thoroughly. The plate was incubated at 37°C for ~10 min to dissolve the insoluble formazan crystals. Absorbance at 490 nm was recorded using Biotek ELx800 microplate reader (Ontario, Canada). Cell viability was quantified based on the absorbance ratio between treated and control conditions.
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