Vibrant mtt cell proliferation assay kit

The Vibrant MTT cell proliferation assay kit (Life Technologies, Inc. Grand Island, NY, USA) was used for cell viability assay according to the manufacturer’s instruction. Briefly, KSHV-positive, BCBL-1 and BC-3 cells depleted for LANA and NAP1L1, and HEK 293L cells depleted for NAP1L1 (shNAP1L1) and control (shCon) cells were grown in RPMI 1640 and DMEM medium without phenol red. Approximately, 100,000 cells were transferred to each well in a 96 well microplate. A total of 10 μl of the 12 mM MTT (3-[4,5-dimethyl-2- thiazolyl]-2,5-diphenyl tetrazolium bromide) stock solution was added to each well. One hundred microliters of medium alone was used as negative control. Cells were incubated at 37 °C for 4 h followed by addition of 100 μl of the SDS-HCl solution. The microplate was then incubated at 37 °C for another 4 h and the absorbance was recorded at 570 nm. Assays were performed in triplicate.

Growth medium DMEM, fetal bovine serum (FBS), penicillin/streptomycin, trypsin 5FU, and TRI reagent were purchased from Sigma-Aldrich (St. Louis, MO, USA). Vibrant MTT cell proliferation assay kit was purchased from Life Technologies Corporation (Carlsbad, CA, USA), and fluorescein isothiocyanate (FITC) conjugated antibody of integrin α_vβ₃ was purchased from BD Biosciences (San Jose, CA, USA). Xeno Light D-luciferin was purchased from Perkin Elmer (Billerica, MA, USA). PEG conjugated tetraiodothyroacetic acid (P-bi-TAT) was synthesized at the Pharmaceutical Research Institute, as described by Rajabi et al. [28 (link)].

An equal number of cells plated in 96-well plates were treated with DMSO, riluzole, A841720 or Bay 36-7620 for 48 h. After the incubation, cell growth was monitored using the Vibrant MTT Cell Proliferation Assay Kit (Molecular Probes). Briefly, cells were incubated with MTT labeling reagent for 4 h, then the solubilization solution (sodium dodecyl sulfate (SDS) in HCl) for 4 h, and the absorbance was read at 570 nM with a microplate reader.

The proliferation analysis was performed using two different methods: the proliferating cell nuclear antigen (PCNA) staining for PCa cells and the Vibrant MTT Cell Proliferation Assay Kit (Molecular Probes, Eugene, OR) for pancreatic and liver cancer cells. LNCaP and PC-3 cells were grown and treated with 1 μM DGAT1 inhibitor for 24 h on glass coverslips. After the treatments, the cells were washed three times with PBS, fixed in 4% paraformaldehyde for 20 min., and stained with PCNA antibody (1:40 – Cat. No. M0879 - Dako, Denmark). After the staining, the coverslips were mounted on glass slides and the percentage of positive cells in the DNA synthesis phase was determined. A minimum of 50 cells were counted in each group. Regarding PANC-1 and Hepa-1c1c7 cell lines, a total of 10⁴ cells per well were seeded into 96-well culture plates and treated for 24 h with 1 μM DGAT1 inhibitor. After 24 h, the medium was replaced with 100 µl of fresh medium and 10 µl of 12 mM MTT was added to each well. After an incubation of 4 h at 37 °C, 100 µl of SDS-HCL solution was added to solubilize the formazan product for an incubation of 4 h at 37 °C. The optical density was then determined using a spectrophotometer at a wavelength of 570 nm.