Methyl thiazolyl tetrazolium (mtt)

The effect of extracts, fractions and pure compound(s) on cell viability were determined using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay (Applichem A2231). A549, HT29 and WI-38 cells, seeded in 96-well plates, after treatment times (24 and 48 hours), were treated with 10 µl (5 mg ml⁻¹) of MTT and incubated for 3 hours. After the incubation time, isopropanol was used to dissolve purple formazan crystals. The absorbance was recorded on a microplate reader at a wavelength of 570 nm (Multiskan FC, THERMO SCIENTIFIC). The effect on cell viability was evaluated as percent of cell viability calculated as the ratio between mean absorbance of each sample and mean absorbance of controls.

RAW Blue cell line viability was assessed by the mitochondrial metabolisation of the tetrazolium salt 3‐(4,5‐methyltiazol‐2yl‐)‐2,5diphenyl‐tetrazolium bromide (MTT) (AppliChem, Germany) colorimetric assay. Cells were maintained and plated as explained previously. The same treatment used for QuantiBlue assay was performed. Then MTT (0.5 mg·ml⁻¹) was added, and cells were incubated at 37°C for 2 h. Cell media were removed and formazan crystals were dissolved in DMSO. OD was measured at 570 nm (OD₅₇₀) on a microplate spectrophotometer (PherastarFSX, BMG Lab).

Fetal bovine serum (FBS), Williams E medium, dimethylsulfoxide (DMSO), bovine insulin, hydrocortisone hemisulfate, and MDL72.527 were obtained from Sigma (St. Louis, MO, USA), whereas MTT was purchased from Applichem (Darmstadt, Germany). DMEM was from Life Technologies (Carlsbad, CA, USA). Mint reverse transcriptase, qPCRmix-HS and qPCRmix-HS SYBR master mixes were purchased from Evrogen (Moscow, Russia). Unmodified DNA oligonucleotides were synthesized by Lytech (Moscow, Russia) or Evrogen, and Taqman probe was from DNA synthesis (Moscow, Russia). Primary rabbit antibodies to E-cadherin (24E10), N-cadherin (D4R1H), claudin-1 (D5H1D), and ZO-1 (D7D12) were purchased from Cell Signaling (Leiden, Netherlands), whereas primary mouse antibodies to β-actin (ab3280) and anti-rabbit (ab6721) and anti-mouse (ab6728) secondary antibodies conjugated to horseradish peroxidase (HRP) were from Abcam (Cambridge, UK). (R)-3-Methylspermidine (MeSpd) [26 (link)] and (R,R)-1,12-dimethylspermine (Me₂Spm) [27 (link)] were synthesized as described earlier. DENSpm was earlier synthesized as described in [28 (link)]. DFMO was a kind gift of Prof. P. Woster (Medical University of South Carolina, Charleston, SC, USA).

Cytotoxicity was assessed using a dimethyl-thiazol diphenyl tetrazolium bromide (MTT) assay. MTT (AppliChem GmbH, Darmstadt, Germany) assay was performed as previously described [43 (link)]. In MTT assay, the metabolic activity of the cells is monitored. It is a convenient colorimetric assay that measure the activity of Nicotinamide adenine dinucleotide phosphate (NAD(P)H)-dependent cellular oxidoreductase enzymes. There is a correlation between the metabolic activity and the number of viable cells. Cells were treated with 122.4 and different chemotherapeutics drugs for 72 h. Following incubation for 2 h with MTT reagent, plates were read on wavelength 570 nm.

The stable vehicle (NC) or MUC1 transfected Hep2 cells or stable vehicle shRNA (NC shRNA) or MUC1 shRNA transfected TU686 cells were seeded into a medium containing 10 ml of fresh medium at a concentration of 1 × 10³ cells/ml in a 96-well microplate, after 24 h, cells were irradiated with 0, 2, 4, 6, or 8 Gy γ-irradiation. Cells were allowed to grow for an additional 72 h and cell viability was analyzed using the 3-(4,5-Methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT, Applichem) assay, according to the manufacturer’s protocol. The reaction product was measured on a microtiter plate reader (Wallac VICTOR 1420, PerkinElmer, Waltham, USA) with a reference wavelength of 490 nm.

The viability of transfected HEK293 cells was verified by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, AppliChem, Darmstadt, Germany) assay. Therefore, 150.000 HEK293 cells per well were seeded in a 12-well plate and transfected with different concentrations of CD39 mRNA. After 24 h and 48 h the cells were washed with RPMI (without phenol red, life technologies), trypsinized and 50.000 cells of each sample were incubated with 250 μg MTT dissolved in RPMI (without phenol red) for 4 h at 37°C. Afterwards, the MTT solution was removed and the cells were incubated with 300 μl DMSO (dimethyl sulfoxide, Serva, Heidelberg, Germany) for 10 min at 37°C. The absorbance was measured at 540 nm with the Mithras LB 940 Microplate Reader (Berthold technologies, Bad Wildbad, Germany).