To measure the mitochondrial activity and membrane damage in HEK293 cells, 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT) (MTT Formazan powder; Sigma-Aldrich, Italy) and lactate dehydrogenase (LDH) (LDH Cytotoxicity Detection KitPLUS; Sigma-Aldrich, Italy) assays were performed following the manufacturer's instructions as in our previous work [24] (link). To create conditions of oxidative stress, cells were incubated for 30 or 60 min either with medium or with medium plus 15 mM tert‑butyl hydroperoxide (tBHP; Sigma-Aldrich, Italy). Absorbance and emission were measured with a multi-label spectrophotometer (VICTOR3, Perkin Elmer, USA) at 570 nm and 490 nm, respectively.
Mtt formazan powder
Manufactured by Merck Group
Sourced in United States
MTT Formazan powder is a chemical compound used in various cell-based assays. It is a purple crystalline powder that is formed when the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is reduced by the mitochondrial enzymes of living cells. The core function of MTT Formazan powder is to serve as a colorimetric indicator for measuring cell viability, proliferation, and cytotoxicity in in vitro experiments.
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Lab products found in correlation
Victor3, by PerkinElmer (1 mentions)
R2408, by Merck Group (1 mentions)
Rosiglitazone, by Merck Group (1 mentions)
Dmem high glucose, by Merck Group (1 mentions)
Normal glucose dmem, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Rnase a, by Thermo Fisher Scientific (1 mentions)
F6627, by Merck Group (1 mentions)
Polarstar microplate reader, by BMG Labtech (1 mentions)
Ir 780 iodide, by Merck Group (1 mentions)
Live dead cell double staining kit, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Pentasodium tripolyphosphate, by Merck Group (1 mentions)
Lb agar, by Merck Group (1 mentions)
Brain heart infusion broth, by Merck Group (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Luria bertani broth, by Merck Group (1 mentions)
5 protocols using mtt formazan powder
1
Mitochondrial Stress Assays in HEK293
2
Cytotoxicity Assay of 5-FU and Rosiglitazone
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Blank glucose Dulbecco's Modified Eagle Medium (DMEM) (Cat. no.: 11966) and normal glucose DMEM (Cat. no: 11885) were purchased from ThermoFisher. High glucose DMEM (D5671 Sigma) was complemented with 4.0 mM L-glutamine, 1.0 mM sodium pyruvate, 10 % fetal bovine serum, 1 % penicillin streptomycin and 1 % amphoterin B. The stock solutions of 5-FU (F6627 Sigma) and rosiglitazone (R2408 Sigma) were prepared in phosphate-buffered saline (PBS) and dimethyl sulfoxide (DMSO) with a concentration of 5 mg/ml and 1 mg/ml respectively. MTT formazan powder (M5655 Sigma) was dissolved in PBS and diluted to 5 mg/ml. Propidium iodide (PI, Cat. no: P1304MP) and RNAse A (12091-021) were obtained from ThermoFisher.
3
MTT Formazan Proliferation Assay
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MTT Formazan powder (Sigma-Aldrich; Cat#88417) was dissolved in sterile PBS at a working concentration of 5 mg/mL. The solution was then filtered using a 0.22 µm syringe filter and stored at 4 °C for short-term and −20 °C for long-term use. U-937 cells were seeded in 96-well plates (2000 cells/well/replicate) after lentiviral transductions. Cells were then incubated with AraC for 72 hr. MTT solution was added at 0.5 mg/mL and incubated overnight prior to the addition of 110 µL of the Colour Development Solution (isopropanol with 0.04 N HCl). Absorbance was recorded with the POLARstar microplate reader (BMG LABTECH; VIC, AUS) at 570 and 630 nm wavelength. Calculations were performed using the equation A570nm–A630nm. Media only control wells were then subtracted from all the readings. The addition of MTT solution and colour development solution was repeated at 0 and 72 h, with plate absorbance readings occurring at the same time point.
4
Evaluation of Cell Viability Reagents
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MnCl2 .4H2O, NaOH and H2O2 were purchased from Kemiou chemical company (Tianjin, China). IR780 iodide, DAPI, MTT Formazan powder and Live/Dead cell double staining kit were all obtained from Sigma-Aldrich Company (Saint Louis, USA). Doxorubicin (DOX, Mw: 543.52 g/mol) was obtained from Meilun Biotechnology, Co. Ltd (Dalian, China). Fetal bovine serum, RPMI-1640 medium, penicillin and streptomycin were all purchased from HyClone (GE, USA).
5
Chitosan-Based Antimicrobial Formulations
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Highly deacetylated chitosan (residual acetylation degree approximately 16 % as determined by means of 1 H-NMR), was purchased from Sigma-Aldrich (Chemical Co. USA). The relative molar mass (''molecular weight'', MW) of chitosan determined by intrinsic viscosity measurements was found to be around 690,000 [24] . Chitlac (lactosemodified chitosan, CAS registry number 85941-43-1) was prepared according to the procedure reported elsewhere starting from highly deacetylated chitosan [24] . The composition of Chitlac was determined by means of 1 H-NMR and resulted to be: glucosamine residue 20 %, N-acetylglucosamine 18 % and 2-(lactit-1-yl)-glucosamine 62 %. The relative MW of Chitlac is around 1.5 9 10 6 . Pentasodium tripolyphosphate (TPP), sodium chloride (NaCl), glycerol (ReagentPlus Ò C 99.0 %), silver nitrate (AgNO 3 ), ascorbic acid (C 6 H 8 O 6 ), MTT formazan powder [1-(4, 5-dimethylthiazol-2-yl)-3,5-diphenylformazan], phenazine methosulfate (PMS) powder and phosphate buffered saline (PBS) were all purchased from Sigma-Aldrich (Chemical Co. USA). Luria-Bertani (LB) broth, LB Agar and Brain Heart Infusion (BHI) broth were from Sigma-Aldrich (Chemical Co. USA). All other chemicals and reagents were of the highest purity grade commercially available.
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