Mtt cell proliferation kit

The MTT cytotoxicity assay is a standard method prior to detailed in vitro study. Equal sizes, weights, and volumes (5 mm × 5 mm × 5 mm), (0.3 g), (300 µL)) of untreated and treated hydrogel scaffold materials in triplicate were incubated in 2 mL Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Sigma-Aldrich) with 10% v/v of fetal bovine serum in 24 well cell culture plates at 37 °C, in 5% CO₂ and fully humidified air for 1, 3, 5, 7, 10, and 14 days. The liquid extraction medium (10 µL each) were collected for MTT assay. Here, we used an indirect method of measurement to assess cell viability. The L929 mouse fibroblast cell line (ATCC (NCTC clone 929; L cell, L-929 derivative of Strain L; ATCC CCL-1)) with an initial density of 5,000 cells/cm² per well at a volume 100 µL were seeded in 96-well plates and were cultured for 24–48 h for proper attachment and growth. Then, periodically collected liquid extracts (10 µL each) were added to each well and incubated for 24 h. Finally, the cell metabolic activity was determined using a MTT cell proliferation kit (Sigma-Aldrich, Gibco, St. Louis, MO, USA). Further, the microplsma treated hydrogel system was compared with traditional genipin crosslinked gelatin hydrogel as control group for cytotocity assay by MTT assay with same experimental conditions as mentioned previously for untreated and microplasma treated gelatin hydrogel.

MTT Cell Proliferation Kit (Sigma), a colorimetric method, was used for this test to evaluate the damage that peptides can cause in eukaryotic cells and all cell lines used in this study have been deposited in a collection of the Research Laboratory of Acibadem Mehmet Ali Aydinlar University. The cell lines of 3T3 (ATCC CRL-1658) and HaCaT (ATCC PCS-200-011) were revived under appropriate culture conditions and made ready for the experiment. The final concentration of peptides reached 64 to 0.5 μg/mL. Samples were worked in triplicates for each concentration. Cells incubated without peptide-treatment under the same conditions were used as a control. Magainin II, a known natural AMP that has no cytotoxic effects, was used as a positive control (Bacalum and Radu 2015 (link)). The results were analyzed by reading them with a 96-well plate reader at 550 and 690 nm.

Cell viability and cytotoxicity were assessed using MTT Cell Proliferation Kit (Sigma-Aldrich) and Cytotoxicity Detection kit assays (Roche Diagnostics, Lewes, UK), respectively, according to the manufacturer’s instructions. A microplate reader (SpectraMax 340PC; Molecular Devices Corporation, Sunnyvale, CA, USA) was used and the data were analyzed using the Softmax Pro software (Molecular Devices Corporation).

Cells in triplicate (plated at density of 5000 cells per well) were grown in growth media in the presence or absence of bupivacaine for 24 hrs or as indicated in 96-well format and subjected to MTT assays. The yellow tetrazolium salt MTT was reduced to formazan by intracellular NADPH-oxidoreductases. The formazan crystals were solubilized and quantified by spectrophotometry using plate reader (Epoch Biotech, USA). Assays were performed using an MTT cell proliferation kit (Sigma Aldrich, USA).

To determine the relative cell proliferation, 1.5  ×  10⁴ cells were plated per well in a 96-well plate and cultured for 24 h in complete cell growth media. The next day cells were exposed to PAH- or HC-EVs (50 μg/mL) for 1 h followed by mock-transfection or transfection of miR-486-5p inhibitor or miR-26a-5p mimic for 48–72 h at 37 °C in a 5% CO₂-humidified incubator. Post 72 h of transfection, hPAECs proliferation was determined using MTT cell proliferation kit (Sigma-Aldrich, Stockholm, Sweden AB) following manufacturer’s instructions. Data was expressed as percent proliferating cells with respect to the untreated cells.

The in vitro human serum complement cytotoxicity assays were performed similarly to that described previously [21 (link)]. Briefly, 2 × 10⁵ MSC1 (n = 3), and MSC1-HI (n = 3) cells were plated per well on a 24-well tissue culture plates (Sigma Chemical Co.) and cultured overnight in 1 mL supplemented DMEM media + 10% FBS. Then, 0.5 mL of media was removed per well and the cells were incubated at 37 °C for 1.5 h in one of three groups: negative control group (incubated in 0.5mL of serum-free media), complement group (incubated in 0.5mL of pooled AB human serum containing complement, Innovative Research, Inc, Novi, MI, USA), and positive control group (incubated in 0.5mL of 1% Triton X-100 detergent). Following incubation, all media was removed. Survival of cells was measured using the Cell Proliferation Kit MTT (3-[4, 5-deimethylthiazol-2-yl]-2, 5-deiphenyltetrazolium bromide) assay (Millipore-Sigma, Darmstadt, Germany) as previously described [21 (link)].