Dna prep kit

Trypsin, Leibovitz's L-15 and EMEM medium, fetal bovine serum (FBS), and penicillin/ streptomycin solution (100×) were obtained from Mediatech, Inc. (Herndon, VA, USA). An Annexin V-FITC apoptosis detection kit was obtained from BD Hoffmann-La Roche Inc. (Nutley, NJ, USA). A DNA-prep kit was obtained from Beckman & Coulter (FL, USA). All reagents for RT-PCR and real-time qPCR were obtained from Applied Biosystem (Foster City, CA, USA). Nuclear/cytosol fractionation kit was obtained from BioVision, Inc. (Moutain View, CA, USA). Antibodies against PI3K, phospho-Akt1/2/3 (Ser473), Akt, NFκBp65, pIκBα and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA and Cambridge, UK). All other reagents were purchased from Sigma Chemicals (St Louis, MO, USA). Plasticware was purchased from Falcon Lab (Franklin Lakes, NJ, USA).

7.5 × 10⁴ cells in 100 µl medium were cultured in 96-well plates for 24, 48 or 72 h in the presence or absence of the TLR ligands. Apoptosis was evaluated by measuring annexin-V and propidium iodide (PI) binding using Annexin-V apoptosis detection kit (Santa Cruz Biotechnology). Viable cells were negative for both annexin-V and PI, early apoptotic cells were positive for annexin-V staining, and late apoptotic cells were positive for both annexin-V and PI staining. Cell cycle analysis was performed using DNA-Prep kit (Beckman Coulter) following the protocol recommended by the manufacturer for cell permeabilization and PI staining. The samples were analyzed by flow cytometry using a FACSCanto II flow cytometer (Becton–Dickinson). Samples were run in duplicate with 10,000 events counted per sample.

Size and complexity parameters and cell cycle distribution were analysed by flow cytometry (Cytomics FC500, 1 laser, Beckman Coulter). For that, cells were trypsinized and washed with PBS by centrifugation. Size evaluation was made based on forward scatter and the complexity was evaluated by side scatter. For cell cycle analysis, DNAprep kit (Beckman Coulter) was used. The pellet obtained by centrifugation was resuspended in 50 µL of detergent of the kit, and 1 mL of propidium iodide, incubating 30 min at 37 °C. Cells were maintained at 4 °C in dark until performing the measurement.

The distribution of cell-cycle phases (G₀/G₁, S, and G₂/M) was determined using flow cytometry by measuring the DNA content of nuclei labeled with propidium iodide as described previously [15 (link)]. Briefly, human colorectal (SW1116 and SW837) and breast (HTB 26 and HTB132) cancer cell lines were plated (5 × 10⁵ cells/ml) in 24-well plates and incubated at 37°C in a non-CO₂ incubator for 18 h. The cells were then treated with Nar (3 mM) for 24 h. Untreated and treated human cancer cells were collected by trypsinization, washed with cold phosphate-buffered saline (PBS) and counted. Cells were processed using a DNA-prep kit (Beckman & Coulter) and a DNA-Prep EPICS workstation (Beckman & Coulter). During this process, the cells were treated with a cell-membrane permeabilizing agent (non-ionic detergent) followed by propidium iodide (PI) and RNase. The samples were incubated at room temperature for 15 min before analysis by flow cytometry (FC500, Beckman & Coulter). The percentages of cells in different cell cycle phases were calculated using the Phoenix statistical software package, advanced DNA cell-cycle software (Phoenix Flow System, San Diego, CA).

Cell distribution throughout the cell cycle phases was studied by flow cytometry (Cytomics FC500, 1 laser, Beckman Coulter, Corston, UK). Cells at 24 h after Metf treatments were trypsinized, fixed in 70% ethanol, and washed with PBS by centrifugation. The pellet was resuspended in 50 μL of DNAprep kit (Beckman Coulter) and in 1 mL of propidium iodide and incubated for 30 min at 37 °C.

HCC cells were harvested and fixed in 75% ethanol and stored overnight at 4°C. Then, the cells were stained with a DNA Prep Kit (Beckman Coulter, Brea, CA, USA), and the percentage of cells at different stages was determined by flow cytometry according to DNA content.

Flow cytometry was used to monitor the disruption in cell cycle phases (Go/G1, S and G2/M) by measuring the DNA content of the nuclei labeled with propidium iodide (PI), as previously described [20 (link)]. Briefly, SW1116 and SW837 cells were plated (2.5 × 10⁵ cells/ml) into 24-well plates and incubated at 37 °C in a non-CO₂ incubator. Cells were treated with MF (1.5 mM) for 24 h starting 18 h after seeding the cells in culture. Untreated and MF-treated cells were collected by trypsinization, washed with cold PBS and counted. Cells were processed using a DNA-prep kit (Beckman & Coulter, FL, USA) and a DNA-Prep EPICS workstation (Beckman & Coulter). During this process, cells were treated with a cell-membrane permeabilizing agent followed by a treatment with PI and RNAase followed by incubation at room temperature for 15 min before analysis by flow cytometry (FC500, Beckman & Coulter). The percentage of cells in different cell cycle phases was calculated using the Phoenix statistical software package (Phoenix Flow System, San Diego, CA).