Microtiter plate spectrophotometer

In vitro cytotoxic effects of recombinant NprvNeps was assessed as described previously [69 (link),70 (link)] with slight modifications. A Vero cell line (ECACC 88020401, African Green Monkey kidney), widely used for the assessment of the cytotoxic effects of chemicals, toxins and other substances on mammalian cells at the molecular level [71 (link)], was grown and maintained according to Ammerman et al. [71 (link)]. Microtiter plates were incubated at 37 °C in 5% CO₂ for 24 h. Recombinant NprvNeps in PBS were filtered through a sterile 0.2 µm pore size syringe membrane filter and prepared at 1 µM, 5 µM, 10 µM concentrations. All NprvNep dilutions then were added to Vero cells [1:1 (v/v) in DMEM - Dulbecco’s Modified Eagle Medium] and incubated for 20 h. After cell treatment, the medium was removed by aspiration and 50 µL of DMEM with 10% resazurin (0.1 mg/mL in PBS) was directly added to each well. The microtiter plates were incubated at 37 °C in 5% CO₂ for 3 h. The absorbance was read at 570 and 600 nm in a microtiter plate spectrophotometer (Biotek Synergy, Colmar, France). Phosphate-buffered saline buffer (PBS) was used as control.

Cell death and/or viability induced by different WaF17.12 concentrations (1000, 5000, 10,000 yeasts cells/mL) after 24 h and 48 h was evaluated, using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and PI (Sigma-Aldrich, Saint Louis, U.S.A.) assays.
In MTT assay, HaCaT cells (7.5 × 10⁵ cells/mL) were seeded into 96-well plates and co-cultured with different yeast concentrations or the vehicle (DMEM). Upon treatment, the supernatant was removed, and the cells were washed twice with PBS 1×. MTT (0.8 mg/mL) was added to the samples and incubated for 3 h. Then, the supernatants were discarded, and 100 μL/well DMSO were added to dissolve the formazan crystals. The colored solutions were read by a microtiter plate spectrophotometer (BioTek Instruments, Winooski, VT, U.S.A.). Six replicates were used for each treatment. The experiments were repeated three times.
For the PI assay, HaCaT cells were incubated with 20 µg/mL of PI in 1× PBS, for 30 min at 20 °C. PI penetrates cells with altered membranes intercalating with the broken DNA, a typical process of cell necrosis. Samples were analyzed by a FACScan cytofluorimeter, using the CellQuest software. The experiments were repeated three times.

In vitro cytotoxicity was performed as previously described [13 (link),44 (link)], with slight modifications. Two cell lines were grown and maintained according to Ammerman et al. [45 (link)]: a Vero cell line (ECACC 88020401, African Green Monkey Kidney cells, GMK clone) and a 3T3 cell line (ECACC 86110401, mouse embryonic fibroblasts, A31 clone A31). The microtiter plates were incubated at 37 °C in 5% CO₂ for 24 h. Vero and 3T3 cells were treated, for 20 h, with the crude extracts (1:1 in DMEM-Dulbecco’s Modified Eagle Medium). Four different concentrations of each crude extract were analysed (1, 0.5, 0.1 and 0.05 mg·mL⁻¹ in phosphate buffered saline (PBS)). After the incubation period, the medium was removed by aspiration and 50 µL of DMEM with 10% resazurin (0.1 mg·mL⁻¹ in PBS) was added to each well to assess cell viability. The microtiter plates were incubated at 37 °C in 5% CO₂ during 3 h. The absorbance was read at 570 and 600 nm wavelengths in a microtiter plate spectrophotometer (Biotek Synergy, Portugal). PBS was used as control.

T24 and 5637 BC and A498 RCC cells (8 × 10⁴/mL) were plated in 96-well plates and treated for 24, 48 and 72 h with different doses of CPS (1–500 μM). At the end of the treatment, 0.8 mg/mL MTT was added to the samples and incubated for 3 h. The medium was removed from the wells, the formazan crystals were dissolved with 100 µL/well DMSO and the absorbance was read by a microtiter plate spectrophotometer (BioTek Instruments, Winooski, VT, USA). Four replicates were carried out for each treatment.

Three × 10⁴/mL cells were plated in 96-well plates and treated with different doses of TMZ (125–500 μM), alone or in combination. After that, the samples were incubated for another 3 h with 0.8 mg/mL of MTT. A microtiter plate spectrophotometer (BioTek Instruments, Winooski, VT, USA) was used to measure the color of solutions after the formazan crystals had been dissolved with 100 μL of DMSO per well. Four replicates were used for each treatment. IC₅₀ values correspond to the drug concentration that induces 50% of cell growth inhibition compared to control cells. IC₅₀ values were calculated using GraphPad Prism^® 5.0a (GraphPad Software, San Diego, CA, USA).

Three ×10⁴/mL siTRPML1, siTRPML2 and siTRPML1/TRPML2 GBM cells were plated in 96-well plates for 24 h and 48 h post-transfection. Then, 0.8 mg/mL of MTT was added to the samples and incubated for an additional 3 h. After the removal of medium from the wells, the formazan crystals were dissolved with 100 µL per well of DMSO and the colored solutions were read by a microtiter plate spectrophotometer (BioTek Instruments, Winooski, VT, USA). Four replicates were used for each treatment.