Tacs mtt cell proliferation assay

Neuronal viability was assessed using a MTT assay kit (TACS MTT Cell Proliferation Assays, #4890-25-K, Trevigen) according to the manufacturer's instructions with minor modifications. Mouse primary neurons were incubated with HMW or LMW SEC fractions from rTg4510 brain extracts (12-months-old, PBS-3,000g, 10 ng ml⁻¹ human tau) in 96-well plate (Corning, #3603) (2.5 × 10⁴ cells/well in 100 μl) for 48 h. MTT (10 μl) was added to each well at 48 h and neurons were incubated for 2 h in an incubator (37 °C in 5% CO₂). When purple precipitate is visible under the microscope, 100 μl of detergent reagent was added to each well and incubated for 4 h at R.T. The absorbance of each well was measured at 600 nm in a microplate reader (Wallac Victor 1420 Multilabel Counter, Perkin Elmer).

Cell viability and proliferation of the canine OS cell lines (Abrams, HMPOS, GD-17 and Gracie) treated with tRNA/miR34-a was determined using a MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) based-colorimetric assay (TACS MTT Cell Proliferation Assay, Trevigen, Gaithersburg, MD), according to manufacturer’s instructions. Briefly, canine OS cells were seeded in 96-well cell culture microplates (Corning Costar, Corning, NY). After 24 hours, cells were treated with tRNA/miR-34a or tRNA/MSA (0, 1, 5, 10, 20 and 30 nM) in triplicate by means of jetPRIME transfection reagent (Polyplus-transfection SA, New York, NY). After 8 hours, transfection mixture was replaced with supplemented RPMI 1640 medium. MTT assays were performed 48 hours after treatment. Cells were incubated with MTT reagent in the dark at 37°C for 2 hours. The detergent reagent was then added to all wells, and plates were returned to the dark at 37°C for other additional 2 hours. Following the incubation, absorbance was measured at 570 and 690 nm employing a microplate spectrophotometer (SpectraMax 190, Molecular Devices LLC., Sunnyvale, CA). IC₅₀ and Hill Slope values were calculated using the non-linear regression inhibitory concentration versus normalized analysis with variable slope implemented in GraphPad Prism v7.04 (GraphPad Software, La Jolla, CA).

To test whether Rh2 directly kills cells at the selected concentrations, TACS MTT cell proliferation assay (Trevigen, Gaithersburg, MD, USA) was conducted to evaluate cell viability according to the manufactory’s instruction, as we described [23 (link)]. Briefly, 3T3-L1 cells were seeded in 12-well plates (about 20% confluent) with different dosages of Rh2 from 30 μM to 300 μM for 72 h. Cells were then incubated with MTT reagent (100 μL per well) for 4 h and added detergent reagent (500 μL each well) and incubated in the dark for 4 h. The relative cell viability was determined by the absorbance at 570 nm using a Synergy H1 hybrid reader (BioTek Instruments, Inc. Winooski, VT).