Mts cell proliferation assay

Manufactured by Promega

Sourced in United States

The MTS cell proliferation assay is a colorimetric method for determining the number of viable cells in proliferation. It utilizes a tetrazolium compound that is bioreduced by metabolically active cells to form a colored formazan product, which can be quantified by measuring the absorbance.

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36 protocols using mts cell proliferation assay

Evaluating Temodar's Efficacy in GBM

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TMZ (Temodar, Schering Plough) was used to investigate the effects of GBM drug treatment. To determine the effective dose (ED₅₀) of the drug, cells were seeded at a density of 2,000 cells per well in a 96-well plate overnight, and treated with the drug (TMZ) or vehicle (final concentration of 0.1% DMSO) for 4 days. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) cell proliferation assay (Promega). For cellular and exosome analyses of drug effects, GBM cells were given a dosage of TMZ below their respective ED₅₀ concentrations (∼10% ED₅₀) before subsequent promoter methylation sequencing and mRNA analyses.

Shao H., Chung J., Lee K., Balaj L., Min C., Carter B.S., Hochberg F.H., Breakefield X.O., Lee H, & Weissleder R. (2015). Chip-based analysis of exosomal mRNA mediating drug resistance in glioblastoma. Nature Communications, 6, 6999.

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Assessing Nanoparticle Neutralization of Cytotoxic Fractions

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To assess the ability of MΦ-NPs to neutralize the biological activity of the PaS-1 fraction, 200 μg of the nanoparticles was incubated with either 20 μg or 40 μg of PaS-1 in water at 37 °C for 15 min, followed by adjustment to 1× PBS in a total volume of 100 μL. To test for hemolytic activity, an equal volume of 2.5% mouse RBCs was added, and the degree of hemolysis was measured after 30 min as described above. To test the impact of MΦ-NP preincubation on cytotoxic activity, J774 cells were plated into 96-well plates at a cell density of 6,000 cells per well. After allowing 24 h for attachment, the cells were incubated with PaS-1 at 20 μg/mL or 40 μg/mL and MΦ-NP at 200 μg/mL for 72 h. Cell viability was assayed using an MTS cell proliferation assay (Promega) following the manufacturer’s instructions. Untreated cells were used as the 100% viability control. For both studies, equivalent amounts of PaS-1 only or MΦ-NPs only were used for comparison. The impact of nanoparticle preincubation on the cytotoxic activity of the PaS-2 fraction was tested in a similar manner, with 200 μg/mL of the MΦ-NPs incubated with either 1 μg/mL or 4 μg/mL of PaS-2. Lack of hemolytic activity was evaluated by incubating with RBCs at a final PaS-2 protein concentration of 200 μg/mL.

Wei X., Ran D., Campeau A., Xiao C., Zhou J., Dehaini D., Jiang Y., Kroll A.V., Zhang Q., Gao W., Gonzalez D.J., Fang R.H, & Zhang L. (2019). Multiantigenic Nanotoxoids for Antivirulence Vaccination against Antibiotic-Resistant Gram-Negative Bacteria. Nano letters, 19(7), 4760-4769.

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MTS Assay for Cell Viability Evaluation

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3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell proliferation assay (Promega Corporation, Fitchburg, WI, USA) was used to determine cell viability. Briefly, cells were cultured to a confluence of 90% in 75 cm² cell culture flasks. Then the cells were passed into 96-well plates (7,500 cells/well). Each well contained 100 μL of culture medium in the presence or absence of various concentrations of drugs or dimethyl sulfoxide as a control. After incubation for 24, 48, or 72 hours, 20 μL of the MTS reagent was added to each well. The plates were then put back to the incubator for an additional 2 hours. The optical density was then determined at 492 nm using a microplate reader (LabSystems Multiskan MS). Experiments were conducted in triplicate and repeated at least three times. The half maximal inhibitory concentration (IC50) values were defined as the concentration of drug producing 50% inhibition of cell growth. The resistance index corresponded to the ratio of IC50 values between the resistant and parental cell lines.14 (link)

Huang L., Hu C., Di Benedetto M., Varin R., Liu J., Wang L., Vannier J.P., Jin J., Janin A., Lu H, & Li H. (2014). Induction of multiple drug resistance in HMEC-1 endothelial cells after long-term exposure to sunitinib. OncoTargets and therapy, 7, 2249-2255.

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Quantifying Cell Proliferation with MTS Assay

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The MTS cell proliferation assay (Promega) was performed according to the manufacturer’s instructions. Briefly, cells were seeded at 8,000 cells (in 100 µL medium) per well into 96-well plates, incubated overnight and exposed to treatments for the indicated time periods. Then 20 µL of CellTiter® 96 Aqueous One So lution Reagent was added into each well. After 4 h incubation at 37°C, the quantity of forma zan product was measured by recording the absorbance at 490 nm with a 96-well plate reader (SpectraMax M5; Molecular Devices). Cell viability was calculated as a percentage of the control group (normalized to 100%).

Gao Y., Zhou S., Xu Y., Sheng S., Qian S.Y, & Huo X. (2018). Nitric Oxide Synthase Inhibitors 1400W and L-NIO Inhibit Angiogenesis Pathway of Colorectal Cancer. Nitric oxide : biology and chemistry, 83, 33-39.

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Evaluating Cell Viability with MTS Assay

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CP70 and SKOV3 cell viabilities were detected using the MTS cell proliferation assay (Promega). Briefly, CP70 and SKOV3 (1000 cells each) were seeded in 96-well plates containing 100 μL media/well overnight. Then, they were exposed to nano-NI or niclosamide for 72 h. The MTS solution was prepared from the CellTiter 96R aqueous MTS reagent powder and kept at −20°C for long-term storage, and at 4°C away from light before use. Next, 20 μL of the MTS solution was added to each well, where the viable cells generated soluble formazan. After an incubation period of 45 to 60 min, the fluorescence absorbance at 490 nm was measured using a 96-well microplate reader (BioTek). Each reaction was assayed at least thrice. The results are expressed as the ratio of the absorbance of each sample to that of the untreated samples with media alone. The proliferation assays were performed in triplicate. All in vitro studies were conducted in triplicate in two independent experiments in the different cell lines.

Lin C.K., Bai M.Y., Hu T.M., Wang Y.C., Chao T.K., Weng S.J., Huang R.L., Su P.H, & Lai H.C. (2016). Preclinical evaluation of a nanoformulated antihelminthic, niclosamide, in ovarian cancer. Oncotarget, 7(8), 8993-9006.

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Photochemical Hydrogel Encapsulation

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Irgacure
2959 (2-hydroxy-1-[4-(2-hydroxyethoxy)
phenyl]-2-methyl-1-propanone; I-2959) was kindly provided by Ciba
Specialty Chemicals (Basel, Switzerland). PEGDA 700, alginic acid
sodium salt from brown algae (low viscosity), glycerol, paraformaldehyde,
ethanol, bisbenzimide H 33258, Dulbecco’s phosphate buffered
saline (DPBS) with CaCl₂ and MgCl₂, fetal bovine
serum (FBS), calcium chloride hexahydrate, sodium azide, and irinotecan
hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO). Fluorescence
activated cell sorter (FACS) buffer was prepared as 1% PBS, 5% FBS,
and 0.05% 3 M NaN₃. Human U251 malignant glioma (U251 MG)
cells were a kind gift from Dr. Lena Al-Harthi at Rush University
and were cultured in DMEM (Corning, NY) supplemented with 5% FBS.
HUVECs and EBM-2 medium along with supplements and growth factors
for the cells were purchased from Lonza (Walkersville, MD). Alexa
Fluor 647 conjugated mouse antihuman CD309 (VEGFR-2) antibody and
Alexa Fluor 647 mouse IgG1 (κ isotype control; FC) antibody
were purchased from Biolegend (San Diego, CA). MTS cell proliferation
assay was purchased from Promega (Madison, WI). Water used in all
experiments was deionized to 18.2 MΩ·cm (Nanopure II, Barnstead,
Dubuque, IA). All chemicals were purchased at standard grades and
used as received.

Sharma V., Köllmer M., Szymusiak M., Nitsche L.C., Gemeinhart R.A, & Liu Y. (2014). Toroidal-Spiral Particles for Codelivery of Anti-VEGFR-2 Antibody and Irinotecan: A Potential Implant to Hinder Recurrence of Glioblastoma Multiforme. Biomacromolecules, 15(3), 756-762.

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Cell Viability Assay Protocol

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Cell viability was determined by fluorescence measurement as previously described.^{11 (link)} Briefly, cells were seeded in Corning black wall, 96-well plates (VWR, Suwanee, GA), treated with indicated agents in a final volume of 0.2 ml RPMI-1640 complete medium, and incubated at 37°C, 5% CO₂, for the times indicated. Viability was determined using PI as follows. Cells (positive controls corresponding to 100% cell death) were permeabilized by addition of 10 μl of 1 mg/ml digitonin and incubated at 37°C, 5% CO₂ for 20 min, followed by addition of PI dissolved in PBS, at a final concentration of 5 μM. The plate was incubated for 20 min, and viability was calculated as the mean (n = 6) fluorescence (minus non-permeabilized control) at 530 nm excitation and 620 nm emission, using a BIO-TEK Synergy H1 microplate reader (Winooski, VT). Viability was also determined using the MTS Cell Proliferation Assay (Promega), as well as trypan blue (0.4%) exclusion and cell counting on a Countess II apparatus, Life Science Technologies, using disposable hemocytometers, from Invitrogen, Thermo/Fisher Scientific; cell growth rates were assessed by the same method.

Fisher-Wellman K.H., Hagen J.T., Kassai M., Kao L.P., Nelson M.A., McLaughlin K.L., Coalson H.S., Fox T.E., Tan S.F., Feith D.J., Kester M., Loughran TP J.r., Claxton D.F, & Cabot M.C. (2022). Alterations in sphingolipid composition and mitochondrial bioenergetics represent synergistic therapeutic vulnerabilities linked to multidrug resistance in leukemia. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 36(1), e22094.

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Cell Viability Assay Using MTS

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The MTS cell proliferation assay (Promega, Southampton, UK) was performed according to the manufacturer’s instructions. Briefly, cells were seeded at 8000 cells (in 100 μL medium) per well into 96-well plates, incubated overnight and exposed to treatments for the indicated time periods. Then 20 μL of CellTiter ® 96 Aqueous One Solution Reagent was added to each well. After 4-h incubation at 37 °C, the quantity of formazan product was measured by recording the absorbance at 490 nm with a 96-well plate reader (SpectraMax M5; Molecular Devices). Cell viability was calculated as a percentage of the control group (normalized to 100%).

Gao Y., Zhou S., Pang L., Yang J., Li H.J., Huo X, & Qian S.Y. (2019). Celastrol suppresses nitric oxide synthases and the angiogenesis pathway in colorectal cancer. Free radical research, 53(3), 324-334.

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Virus Killing and Cell Viability Assays

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For the virus killing and cell viability assays, cells in 96 well plates were exposed to either the VVNIS-W or VVNIS-C at specified multiplicity of infection (MOI; 0, 0.001, 0.01, 0.1, 1, and 10). Cell viability was assessed at 72 hours post infection using the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell proliferation assay according to manufacturer’s instructions (Promega, Madison, WI). Photos were taken using a Nikon Eclipse TE300 microscope mounted with a Nikon U2 digital camera prior to the MTS assay. The median effective concentration (EC₅₀) values of the viruses in each cell line were calculated using PRISM analysis software (GraphPad, La Jolla, CA).
For the viral progeny propagation assays, cells (2×10⁵/well) were seeded in 12 well plates, infected with viruses (MOI 0.02) and incubated at 37°C. Two hours later, the virus inoculum was removed and replaced with growth media. Cells were harvested at 48 hours post virus infection, and viral titers were determined by TCID₅₀ assay on HeLa cells.

Liu Y.P., Wang J., Avanzato V.A., Bakkum-Gamez J.N., Russell S.J., Bell J.C, & Peng K.W. (2014). Oncolytic Vaccinia Virotherapy for Endometrial Cancer. Gynecologic oncology, 132(3), 722-729.

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Cisplatin Sensitivity in RIT1-Knockdown Cells

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We seeded KYSE150 and ECa109 cells with RIT1-knocked-down cells (RIT1 sh) in 96-well plates at a density of 1.5 × 10³ cells per well and gave treatment with cisplatin (CDDP) at different concentrations for 72 h. Cell viability was determined by MTS Cell Proliferation Assay (Promega, Madison, WI, USA). The experiments were repeated for three times.

Feng Y.F., Lei Y.Y., Lu J.B., Xi S.Y., Zhang Y., Huang Q.T., Wu Q.L, & Wang F. (2018). RIT1 suppresses esophageal squamous cell carcinoma growth and metastasis and predicts good prognosis. Cell Death & Disease, 9(11), 1085.

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