Celltiter 96 aqueous one solution assay

Manufactured by Promega

Sourced in United States, Poland

The CellTiter 96 AQueous One Solution Assay is a colorimetric method for determining the number of viable cells in proliferation or cytotoxicity assays. The assay measures the metabolic activity of cells, which is directly proportional to the number of living cells in the culture.

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Lab products found in correlation

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CellTiter 96 (150 citations) CellTiter 96 AQueous (88 citations)

100 protocols using celltiter 96 aqueous one solution assay

Silencing SNAIL in Rhabdomyosarcoma Cells

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RH30, RH41 cells and human myoblasts were transfected with 20 nM siRNA against SNAIL (combination of two Silencer Select siRNA ID variants: s13185 and s13187, Ambion Inc., Austin, TX, USA) or scrambled control siRNA (Silencer Select Negative Control #1 siRNA, cat. 4390844, Ambion) using Lipofectamine 2000 (Invitrogen) or Lipofectamine RNAiMAX transfection reagent according to vendor’s instructions. Twenty-four hours later, the transduction medium was changed to differentiating medium supplemented with 2% HS. RNA or protein was isolated 72 h after transfection. The cellular morphology was visualized using Wright’s stain (Sigma-Aldrich). Proliferation of RH30 and RH41 cells transfected on 96-well plates with siRNA was estimated using CellTiter 96® AQueous One Solution assay (Promega, WI, USA), according to vendor’s protocol.

Skrzypek K., Kusienicka A., Trzyna E., Szewczyk B., Ulman A., Konieczny P., Adamus T., Badyra B., Kortylewski M, & Majka M. (2018). SNAIL is a key regulator of alveolar rhabdomyosarcoma tumor growth and differentiation through repression of MYF5 and MYOD function. Cell Death & Disease, 9(6), 643.

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Tetrazolium-based Cell Viability Assay

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Twenty-four hours after OGD, cell viability was assessed using the tetrazolium-based CellTiter 96 AQ_ueous One Solution Assay (Promega, Madison, WI, USA). Twenty microliters of solution were added to the culture wells. Cultures were incubated for 1 h at 37 °C followed by measurement of absorbance at λ_abs = 492 nm with a FLUOstar OPTIMA microplate reader (BMG Labtech GmbH, Offenburg, Germany). Results were compared with paired cultures exposed to the same neurotoxic stimulus on the same day and cell viability was expressed as the percentage of the corresponding control culture (untreated and not exposed to the lethal insult) using the following formula:

\begin{matrix} % {viability}_{SAMPLE} = ({absorbance}_{SAMPLE} - {absorbacne}_{BACKGROUND}) \times \\ 100 / ({absorbance}_{CONTROL} - {absorbance}_{BACKGROUND}) . \end{matrix}

Dutta S., Rutkai I., Katakam P.V, & Busija D.W. (2015). The mechanistic target of rapamycin (mTOR) pathway and S6 Kinase mediate diazoxide preconditioning in primary rat cortical neurons. Journal of neurochemistry, 134(5), 845-856.

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Assessing Aortic Tissue Viability

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Metabolic activity of aortic tissue was assessed using an assay based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) by viable cells with CellTiter 96^® AQ_ueous One Solution Assay (Promega). Tissue punches were incubated in DMEM overnight at 37 °C. Medium was exchanged before MTS assay was performed according to the manufacturer’s protocol. Absorbance at the 490 nm wavelength was measured (SpectraMax 340PC; Molecular Devices, Sunnyvale, CA, USA).
Necrotic cells or cells with membrane damage in aortic tissue were determined by a lactate dehydrogenase (LDH) release assay (CytoTox-ONE™ Homogeneous Membrane Integrity Assay; Promega). Tissue punches were incubated in DMEM for 1 h at 37 °C before assay was performed according to the manufacturer’s protocol. Fluorescence was read with excitation at 560 nm and emission at 590 nm (Infinite 200 PRO, Tecan, Männedorf, Switzerland).
Caspase 3/7 activity was determined using Caspase-Glo^® 3/7 assay (Promega). Tissue punches were incubated in DMEM for 1 h at 37 °C before assay was performed according to the manufacturer’s protocol. After 1 h incubation, luminescence was measured (Mithras LB 940, Berthold Technologies, Bad Wildbad, Germany).

Schneider M., Stamm C., Brockbank K.G., Stock U.A, & Seifert M. (2017). The choice of cryopreservation method affects immune compatibility of human cardiovascular matrices. Scientific Reports, 7, 17027.

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Evaluating SMS-CTR Cell Proliferation and Mitochondrial Activity

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SMS-CTR cells were seeded on 24-well plates at density of 10 000 cells per well. After 24 hours medium was changed and 24, 48 and 72 hours later cells counted in a Burker hemocytometer chamber. To examine mitochondrial activity of the cells MTT test was done. 2000 of SMS-CTR cells were seeded on 96-well plates. After 24 hours medium was changed for either a medium with 10% FBS or 0.5% BSA and cells were incubated either in normoxia (21% O₂ level) or at 5% O₂ level for 24 hours. Afterwards mitochondrial activity of the cells was estimated with CellTiter 96^® AQueous One Solution assay (Promega), according to vendor's protocol.

Skrzypek K., Kusienicka A., Szewczyk B., Adamus T., Lukasiewicz E., Miekus K, & Majka M. (2015). Constitutive activation of MET signaling impairs myogenic differentiation of rhabdomyosarcoma and promotes its development and progression. Oncotarget, 6(31), 31378-31398.

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Colorimetric Assay for Cellular Toxicity

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Toxicity was determined by a colorimetric assay using a commercially available kit (CellTiter 96^® Aqueous One Solution Assay; Promega, Madison, WI, USA) and following the manufacturer's instructions. Adherent cells were treated with DETC (500 μm, 2 h), and H₂O₂ (50 μm, 30 min) separately, in the absence and presence of tempol (1 mm). Pretreatment of tempol was carried out for 10 min before adding DETC, which remained there with DETC. Cells were suspended in KH buffer and were loaded equally in a 96‐well plate. Subsequently, CellTiter96^® AQ_eous One Solution Reagent (20 μL) was added to each well, and incubated for 2 h at 37 °C and absorbance was read at 490 nm where the resultant color was directly proportional to the number of viable cells.

Saleem M., Pokkunuri I, & Asghar M. (2016). Superoxide increases angiotensin II AT1 receptor function in human kidney‐2 cells. FEBS Open Bio, 6(12), 1273-1284.

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Chd1l Knockdown Impacts Mouse SSC Proliferation

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Mouse SSCs infected with scrambled control or Chd1l shRNA-expression lentiviruses were seeded at a density of 2000 cells per well in 96-well microtiter plates. After 24, 48, 72, and 96 h of culture, proliferation assays were performed using the CellTiter 96® Aqueous One Solution Assay (Promega, Madison, WI), according to the manufacturer's instructions.

Liu S.S., Bai Y.S., Feng L., Dong W.W., Li Y., Xu L.P, & Ma N.F. (2016). Identification of CHD1L as an Important Regulator for Spermatogonial Stem Cell Survival and Self-Renewal. Stem Cells International, 2016, 4069543.

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Cell Viability Assay with Compounds

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Cells were seeded in 96-well
plates (Corning 3598) at 5000 cells/well and treated with indicated
compounds 24 h after plating. Compounds are added at the same time
in cotreatments. Cell viability was assessed using CellTiter 96 Aqueous
One Solution Assay (Promega) according to the manufacturer’s
instructions. Viability was reported as percentages relative to vehicle
control, and EC₅₀ curves were generated with GraphPad Prism
(version 9).

Do Q., Zhang R., Hooper G, & Xu L. (2023). Differential Contributions of Distinct Free Radical Peroxidation Mechanisms to the Induction of Ferroptosis. JACS Au, 3(4), 1100-1117.

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Cell Proliferation, Apoptosis, and Protein Quantification

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Cell proliferation was measured using CellTiter 96 Aqueous One solution assay (Promega Corporation, Madison, WI) according to the manufacturer's directions as described previously [44 (link), 47 (link)]. Cellular apoptosis was measured using APC-Annexin-V assay (BD Biosciences, San Diego, CA). Levels of p62 were measured using p62 ELISA kit according to the manufacturer's instructions (Enzo life Science, Farmingdale, NY). Immunoblot analysis and Real-Time PCR were conducted as previously described [24 , 44 (link), 47 (link)]. Primers used in real-time PCR are shown in table 2.

Gong J., Muñoz A.R., Chan D., Ghosh R, & Kumar A.P. (2014). STAT3 down regulates LC3 to inhibit autophagy and pancreatic cancer cell growth. Oncotarget, 5(9), 2529-2541.

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Monitoring MDA-MB-231 Cell Proliferation

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Proliferation of MDA-MB-231 cells and the NI and I shA1_3 clones during serum starvation was monitored using the MTS assay. 5000 cells were seeded into 96-well plate and left grown for 72 h. Cell viability was determined by CellTiter 96 Aqueous One Solution Assay (Promega) according to manufacturer instructions. Briefly, after 0, 24, 48, 72, or 96 h of incubation in the growth medium with or without serum, 120 μL of MTS solution (MTS reagent diluted 1:6 in PBS containing glucose 4,5 g/L) was added into each well, the plate was incubated for 2.5 h at 37 °C in 5% CO₂ atmosphere, and then absorbance at 490 nm was recorded with a 96-well plate ELISA reader. The same cell viability assay was performed on MDA-MB-468 cells treated with enriched glycoproteins (0.35 µg/well) obtained from MDA-MB-231_shA1_3 expressing or not expressing HMGA1 (NI and I, respectively). Cells were treated after 24 h from seeding.

Resmini G., Rizzo S., Franchin C., Zanin R., Penzo C., Pegoraro S., Ciani Y., Piazza S., Arrigoni G., Sgarra R, & Manfioletti G. (2017). HMGA1 regulates the Plasminogen activation system in the secretome of breast cancer cells. Scientific Reports, 7, 11768.

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MSC Cytocompatibility on Engineered PCL Scaffolds

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To confirm the cytocompatibility character of the etched MEW PCL mesh and engineered membranes, 1×10⁶ and 2×10⁵ MSCs were seeded per sample, respectively and cell proliferation was evaluated using MTS assay (CellTiter 96 AQueous One Solution Assay, Promega Corporation, Madison, WI, USA). At predetermined time points, the cells were incubated for 2 h and absorbance was measured in a microplate reader at 490 nm (Spectra iD3; Molecular Devices, LLC, San Jose, CA, USA). For fluorescence, the cells were fixed with 4% PFA for 30 min at RT, followed by permeabilization with 0.1% Triton X-100 in PB for 5 min at RT. The cells were then washed with PBS and blocked using 1% BSA for 30 min, followed by staining with TRITC-conjugated phalloidin and DAPI (1:1200, Millipore Sigma Company, Burlington, MA, USA) for 1 h according to the manufacturer’s instructions; samples were imaged using an upright fluorescence microscope (Carl Zeiss). SEM was done to observe cell morphology at day 5 on the membranes’ surface. The cells were fixed with 4% paraformaldehyde for 30 min, followed by washing with PBS (2×) for 5 min each. Samples were imaged using a low vacuum Tescan Rise electron microscope (Tescan USA, Inc., Warrendale, PA, USA).

Dubey N., Ferreira J.A., Daghrery A., Aytac Z., Malda J., Bhaduri S.B, & Bottino M.C. (2020). Highly Tunable Bioactive Fiber-Reinforced Hydrogel for Guided Bone Regeneration. Acta biomaterialia, 113, 164-176.

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