Mtt based in vitro toxicology assay kit

Three different sets of human MSC cell line were evaluated in this study, one acquired from Lonza (Allendale, NJ, USA) and two others isolated and expanded from patient bone marrow in our laboratory. Cell proliferation assay of hMSCs grown in various hepatogenic culture conditions reveals the overall impact of sera components (particularly bilirubin) on cell growth. Therefore, we evaluated the in vitro cytotoxic effect of different concentrations of patient sera (10% vs. 5% in DMEM) upon proliferation potential of MSCs. As a control, the impact of normal volunteer donor’s sera (10% and 5% in DMEM) on hMSC proliferation was evaluated. Cells grown in 10% FBS was used as positive control. Cell death assay was performed by annexin V-FITC/PI apoptosis detection kit (Cell Signaling Technology) as per manufacturer’s protocol. Cell proliferation assay was done by direct cell counting using trypan blue on every 3^rd day until 9 days and also by MTT based in vitro toxicology assay kit (Sigma) as per manufacturer’s protocol. An increase or decrease in cell number results in a concomitant change in the amount of formazan formed as measured spectrophotometrically, indicating the degree of cytotoxicity caused by the serum components.

Cells at passage 3 were grown to 2 days post-confluence before being treated with 1 μM dexamethasone, in the absence or presence of 10 μM vanadate, for 7 days. For crystal violet (CV) staining, samples were fixed in 70% ethanol, stained with 0.1% CV for 5 min, and washed three times with PBS before images were captured as described above (see MSC Differentiation and Cytological Staining). Crystal violet was then extracted with 75% ethanol and the absorbance measured at 570 nm. The MTT assay protocol was adapted from the MTT-based in vitro toxicology assay kit from Sigma-Aldrich. Cells at passage 3 were seeded into 96-well plates and treatments commenced at 2 days post-confluence. Cells were treated for 7 days, after which 10 μl of 5 mg/ml MTT stock solution was added to each well and incubated for 2 h. The color reaction was stopped by the addition of 100 μl of 10% Triton X-100 plus 0.1N HCL in isopropanol, and samples were incubated on a plate shaker until the color product was completely dissolved. The color development was quantified spectrophotometrically at 570 nm, and background absorbance at 690 nm was subtracted from each well.

Cell cytotoxicity reagent (MTT based-In Vitro Toxicology Assay Kit), gossypol, LPS and other chemicals (chloroform, DMSO, ethanol, hexane, sodium bicarbonate, sulfuric acid) were from Sigma (St. Louis, MO). Tissue culture reagents (DMEM, MEM, fetal bovine serum, penicillin, streptomycin, L-glutamine) were from Gibco BRL (Thermo Fisher). Tissue culture incubator was water jacket CO₂ incubator, Forma Series II, Model 3100 Series (Thermo Fisher). Tissue culture workstation was Logic + A2 hood (Labconco, Kansas City, MO). Tissue culture plastic ware (flasks, plates, cell scraper) was from CytoOne (USA Scientific, Ocala, FL). Cell counting reagent (trypsin blue dye), slides (dual chamber), counter (TC20 Automatic Cell Counter) and microscope (Zoe Florescent Cell Imager) were from Bio-Rad (Hercules, CA). Molecular sieves were from USA Standard Testing Sieves (Milwaukee, WI). Microplate spectrophotometer (Epoch) was from BioTek Instruments (Winooski, VT).