Enhanced apoptotic dna ladder detection kit

PC, Chol, DMPG, chloroform, and Triton X-100 were supplied from Sigma-Aldrich (Saint Louis, USA). Sephadex-G-50 was acquired from Pharmacia Fine Chemicals (Uppsala, Sweden). Gemcitabine was purchased from Lilly (France), and oxaliplatin was purchased from Sigma (Saint Louis, USA). Fe₃O₄ (10 nm) particles weresupplied from Southwest Institute of Applied Magnetics of China (Chengdu, China). MTT cell proliferation assay kits were received from ATCC (Manassas, USA). Enhanced Apoptotic DNA Ladder Detection Kit was purchased from BioVision (Milpitas, CA, USA). A caspase-3 colorimetric assay kit was purchased from BioVision (USA). Trizol reagent, DNase I enzyme, SuperScript® III Reverse Transcriptase, andSYBR Green PCR Master Mix were purchased from Invitrogen (Carlsbad, CA, USA). BCA protein assay kits were acquired from Pierce (Rockford, USA). PVDF membranes were acquired from Bio-Rad Laboratories (Hercules, USA). Antibodies, namely, rabbit anti-human Bcl-2, BAX, survivin, GAPDH, and goat anti-rabbit IgG-HRP, were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). ECL kits were supplied from Amersham Pharmacia Biotech (Piscataway, USA). All other reagents used were of analytical grade.

FMS-1 and FPS-1 cells were seeded in 6-well plates (BD Falcon 353046) at a dose of 5.0×10⁵ cells/well and incubated for 24 h at 37°C in 5 ml of culture medium. Cells were treated by adding etodolac, dissolved and diluted to the desired concentration by 50 µl of 99.5% ethanol (final concentration of ethanol: 0.99% or less), at a final concentration of 0 (control) or 1 mM. At 24, 48, and 72 h after the etodolac treatment, cells were removed using a rubber policeman. For morphological analysis of dead cells, 50 µl of the cell suspension was cytospun on silane-coated slides, which were stained by May-Giemsa staining and observed by two investigators (HH and MH). Suspended cells were washed in ice-cold phosphate-buffered saline (PBS) twice and pelleted by centrifugation. To detect cell death, whether apoptotic or not, we extracted a DNA sample from each cell pellet using the Enhanced Apoptotic DNA Ladder Detection Kit (BioVision, Mountain View, CA, USA) according to the manufacturer’s protocol. Extracted DNA samples were electrophoresed through 1.8% agarose gels and stained with ethidium bromide.