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Tacs mtt cell proliferation assay kit

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The TACS® MTT Cell Proliferation Assay kit is a colorimetric assay that measures the metabolic activity of cells. The assay is based on the reduction of the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to formazan by metabolically active cells. The amount of formazan produced is proportional to the number of viable cells, and can be quantified by measuring the absorbance at a specific wavelength.

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Lab products found in correlation

Imidazole, by Merck Group (1 mentions) Megatran 1.0 transfection reagent, by OriGene (1 mentions) Dynasore, by Bio-Techne (1 mentions) Thioglycollate medium, by BD (1 mentions) Drabkin s reagent, by Merck Group (1 mentions) Fitc antibody labeling kit, by Thermo Fisher Scientific (1 mentions) Non immune mouse igg, by Merck Group (1 mentions) Triton x 114, by Merck Group (1 mentions) Human m csf, by Merck Group (1 mentions) Carbenicillin, by Thermo Fisher Scientific (1 mentions) Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions) Nhs activated sepharose 4 fast flow beads, by GE Healthcare (1 mentions) Redextract n amp tissue pcr kit, by Merck Group (1 mentions) Trizol reagent, by Thermo Fisher Scientific (1 mentions) Sybr premix ex taq 2, by Takara Bio (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Dexamethasone (dex), by Merck Group (1 mentions) Elx800, by Agilent Technologies (1 mentions) 96 well flat bottom plate, by BD (1 mentions) 96 well flat bottom culture plate, by BD (1 mentions)

13 protocols using tacs mtt cell proliferation assay kit

1

Splenocyte Proliferation Assay by MTT

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The splenocyte proliferation rate was measured by 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay. Briefly, splenocytes were seeded in 96-well plates at 2.5×104 cells/well in 100 uL RPMI-1640 media (10% FBS, and 1% Penicillin-Streptomycin) and treated with ConA (1 μg/mL) for 2 days. After incubation, splenocyte proliferation was performed by TACS MTT cell proliferation assay kit (4890-25-K, Trevigen, Gaithersburg, MD, USA) according to the manufacturer's instructions.
Kim S.K., Kwon D.A., Lee H.S., Kim H.K, & Kim W.K. (2019). Preventive Effect of the Herbal Preparation, HemoHIM, on Cisplatin-Induced Immune Suppression. Evidence-based Complementary and Alternative Medicine : eCAM, 2019, 3494806.
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2

MTT Cell Viability Assay Protocol

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MTT assays were performed using the TACS MTT ® Cell Proliferation Assay Kit (Cat#: 4890-25-K, Trevigen, city, state, USA) and following the manufacturer’s instructions. Briefly, 106 cells in 100 µL serum-free medium were dispensed in 96-well plates and incubated at 37 °C for 18 h, with or without CPZ. Later, 10 µL of MTT reagent was added to each well and incubated for 4 h until the purple dye was clearly visible. Then, 100 µL of detergent reagent (Cat#: 4890-25-02) was added to each well and incubated again at 37 °C for 4 h. The absorbance was read at 570 nm wavelength and the percentage of viable cells for each treatment was calculated as compared to viable cells in untreated control.
Sindhu S., Kochumon S., Shenouda S., Wilson A., Al-Mulla F, & Ahmad R. (2019). The Cooperative Induction of CCL4 in Human Monocytic Cells by TNF-α and Palmitate Requires MyD88 and Involves MAPK/NF-κB Signaling Pathways. International Journal of Molecular Sciences, 20(18), 4658.
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3

Haptoglobin Purification and Characterization

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Human haptoglobin (from pooled human plasma, a mixture of 1-1, 2-1, and 2-2 phenotype, catalog 3536), REDExtract-N-Amp Tissue PCR kit (catalog XNATS), Triton X-114, imidazole, monoclonal anti-human haptoglobin antibodies (catalog H6395), nonimmune mouse IgG, Drabkin’s reagents (catalog D5941), human M-CSF, DAPI, and dexamethasone (catalog D2915) were purchased from Sigma-Aldrich. TACS MTT Cell proliferation assay kit was from Trevigen. Trizol reagent was from Invitrogen. The RevertAid First Strand cDNA Synthesis Kit was from Fermentas. SYBR Premix Ex Taq II was from Takara Bio Inc. Thioglycollate medium was purchased from BD Biosciences. Primers for quantitative PCR (qPCR), trypsin-EDTA, and carbenicillin were from Invitrogen. Protein A/G agarose and isopropyl-D-thiogalacto-pyranoside (IPTG) were purchased from Pierce. NHS-activated Sepharose 4 fast flow beads were obtained from GE Healthcare (catalog 17-0906-01). E. coli strain DH5α was from Novagen. Recombinant human CD163 protein, CD163 expression plasmid (SC117495, NM_004244.3), and MegaTran 1.0 transfection reagent were purchased from Origene. Antibodies for human HO-1 (catalog ab52946) and CD163 (catalog MCA1853) were from Abcam and Serotec, respectively. Anti–human CD163-PE antibody was from BioLegend. FITC antibody labeling kit was from Thermo Scientific. Dynasore was from Tocris Bioscience.
Yang H., Wang H., Levine Y.A., Gunasekaran M.K., Wang Y., Addorisio M., Zhu S., Li W., Li J., de Kleijn D.P., Olofsson P.S., Warren H.S., He M., Al-Abed Y., Roth J., Antoine D.J., Chavan S.S., Andersson U, & Tracey K.J. (2016). Identification of CD163 as an antiinflammatory receptor for HMGB1-haptoglobin complexes. JCI Insight, 1(7), e85375.
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4

Cytotoxic Effects of AM Extracts

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AM ethanolic-extract as well as the organic fractions was tested for cytotoxic effects, if any, on cultured HepG2 and HepG2.2.15 cells. Cells were seeded (0.5 × 105 cells/well, in triplicate) in a 96-well flat-bottom plate (Becton-Dickinson Labware) and grown over night. AM total extract and organic fractions were dissolved in dimethyl sulphoxide (DMSO; 100 mg/mL), followed by dilutions in culture media to prepare five doses (0, 25, 50, 100, and 250 μg/mL) of each. The final concentration of DMSO used never exceeded >0.1% and therefore had no cytotoxicity. The culture monolayers were replenished with complete media containing a dose of AM and incubated for 48 h at 37°C followed by MTT assay (TACS MTT Cell Proliferation Assay Kit, Trevigen) as per the manufacturer's instruction. The absorbance/optical density (OD) was recorded at 620 nm in a microplate reader (BioTek, ELx800) and the data analyzed.
Arbab A.H., Parvez M.K., Al-Dosari M.S., Al-Rehaily A.J., Al-Sohaibani M., Zaroug E.E., AlSaid M.S, & Rafatullah S. (2015). Hepatoprotective and Antiviral Efficacy of Acacia mellifera Leaves Fractions against Hepatitis B Virus. BioMed Research International, 2015, 929131.
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5

In Vitro Cytotoxicity Evaluation

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All compounds were first tested for in vitro cytotoxic effects, if any, on cultured HepG2.2.15 cells. Cells were seeded (0.5 × 105 well, in triplicate) in a 96-well flat-bottom culture plate (Becton-Dickinson Labware) and grown over night. Compounds were first dissolved in 50 μl of dimethyl sulfoxide (DMSO) following final preparation in culture media (1 mg/ml). Five doses (2.5, 5, 10, 20 and 50 μg/ml) of each compound were further prepared by diluting in culture media. The final concentration of DMSO used never exceeded >0.1%, and therefore, had no cytotoxicity. RPMI with 0.1% DMSO served as untreated or negative control. The cultures were replenished with media containing a dose of the drug, and incubated for 48 h followed by MTT assay (TACS MTT Cell Proliferation Assay Kit, Trevigen) as per the manufacturer’s manual. The 50% cytotoxic concentrations (CC50) for all compounds were determined using regression curve in Excel software. The experiment was repeated to confirm the reproducibility.
Parvez M.K., Tabish Rehman M., Alam P., Al-Dosari M.S., Alqasoumi S.I, & Alajmi M.F. (2018). Plant-derived antiviral drugs as novel hepatitis B virus inhibitors: Cell culture and molecular docking study. Saudi Pharmaceutical Journal : SPJ, 27(3), 389-400.
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6

EV-mediated Regulation of EWS Cell Proliferation

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EWS cells were seeded in 96 well plates in a complete medium. 24 h later, cells were treated with EVs (15 µg EVs/10,000 cells) in IMDM + 10% EVs-depleted FBS. Cell proliferation was evaluated after 24 h using the TACS® MTT Cell Proliferation Assay kit (Trevigen, Inc., Gaithersburg, MD, USA) according to the manufacturer’s instructions.
Mancarella C., Giusti V., Caldoni G., Laginestra M.A., Parra A., Toracchio L., Giordano G., Roncuzzi L., Piazzi M., Blalock W., Columbaro M., De Feo A, & Scotlandi K. (2023). Extracellular vesicle-associated IGF2BP3 tunes Ewing sarcoma cell migration and affects PI3K/Akt pathway in neighboring cells. Cancer Gene Therapy, 30(9), 1285-1295.
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7

Cytotoxicity Evaluation of Saponin

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Cytotoxicity was measured using the TACS® MTT cell proliferation assay kit (Trevigen, Gaithersburg, MD, USA). Cells were seeded onto 96-well plates and treated with indicated concentrations of saponin for 48 h. Post treatment, MTT (10 µL per well) was added, and the plates were incubated at 37 °C. Dimethyl sulfoxide (DMSO, 100 µL) was added, and the dark blue formazan product was quantified using a microplate reader at 570 nm (with a 690 nm reference filter) (Molecular Device, Sunnyvale, CA, USA). Relative cell viability (%) is expressed as a percentage relative to non-treated control cells43 (link).
Kim M.K., Lee S.K., Park J.H., Lee J.H., Yun B.H., Park J.H., Seo S.K., Cho S, & Choi Y.S. (2017). Ginsenoside Rg3 Decreases Fibrotic and Invasive Nature of Endometriosis by Modulating miRNA-27b: In Vitro and In Vivo Studies. Scientific Reports, 7, 17670.
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8

Caco-2 Cell Viability Assay

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Caco-2 cells were seeded in 96-well plates (10,000 cells/well) and after incubation for 12 h, cells were treated with cetuximab (Merck, Taiwan). Cell viability was then assessed using the TACS MTT cell proliferation assay kit (TREVIGEN, Gaithersburg, MD, USA), according to the manufacturer’s instructions. The dose–response or time-response curves were analyzed using GraphPad Software (Institute for Scientific Information, Philadelphia, PA, USA).
Chen K.F., Yen C.C., Lin J.K., Chen W.S., Yang S.H., Jiang J.K., Lan Y.T., Lin C.C., Yu H.C., Hsu H.M., Lin W.L, & Teng H.W. (2015). Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an independent prognostic marker in wild-type KRAS metastatic colorectal cancer after colorectal liver metastasectomy. BMC Cancer, 15, 301.
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9

Assessing Sensitivity and Motility in Cell Lines

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Sensitivity to OSI-906 was assessed after 72 h of treatment with the TACS® MTT Cell Proliferation Assay kit (Trevigen, Inc., Gaithersburg, MD, USA) according to the manufacturer’s instructions. Cells were seeded into 96-well plates (2,500 cells/well) in IMDM plus 10% FBS. After 24 h, various concentrations of OSI-906 (0.3–10 µM) were added. Motility assay was performed using Transwell chambers (CoStar, Cambridge, MA, USA). 105 cells in IMDM plus 1% FBS were seeded in the upper compartment, and IMDM plus 1% FBS and IGF1 (50 ng/ml) was added to the lower compartment of the chambers. To assess the response to IGF1, 5 × 104 cells were seeded in 24-well plates, serum starved for 24 h and treated with IGF1 (50 ng/ml) for 24 h. Trypan Blue cell count was employed to assess the response to IGF1. For each independent experiment, samples were in duplicate (Trypan Blue) or triplicate (MTT assay; Motility assay).
Mancarella C., Pasello M., Manara M.C., Toracchio L., Sciandra E.F., Picci P, & Scotlandi K. (2018). Insulin-Like Growth Factor 2 mRNA-Binding Protein 3 Influences Sensitivity to Anti-IGF System Agents Through the Translational Regulation of IGF1R. Frontiers in Endocrinology, 9, 178.
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10

Evaluating FUS Knockdown Effects

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SH-SY5Y or HEK293 cells were transfected twice with control or FUS shRNA with an interval of 24 h between transfections. 24 h after the second transfection, transfectants were trypsinized and plated in 96-well plates. Following GO or mock treatment, cell viability was measured with a TACS MTT Cell Proliferation Assay kit (Trevigen), according to the manufacturer’s instructions. Cell proliferation was measured using a microplate reader (BioRad Model 680) at an absorbance of 570 nm. For the clonogenic survival assay, GO treatment was performed at various concentrations, then defined numbers of control or FUS shRNA transfected cells were plated in 6-well plates. After 10–14 days, cells were stained for 30 min with crystal violet (0.1%) and survival fractions were calculated56 (link).
Wang H., Guo W., Mitra J., Hegde P.M., Vandoorne T., Eckelmann B.J., Mitra S., Tomkinson A.E., Van Den Bosch L, & Hegde M.L. (2018). Mutant FUS causes DNA ligation defects to inhibit oxidative damage repair in Amyotrophic Lateral Sclerosis. Nature Communications, 9, 3683.
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