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In a recent study, the RGD nano structured platform was used for the electrochemical determination of cell cycle determination successfully as shown in Fig. 12 [12].
After assay completion, Hoechst 33342 was added for cell cycle determination and immediately processed on the flow cytometer.
We thank Javier Godino from the Cell Separation Service of the Instituto Aragonés de Ciencias de la Salud (I + CS) for his technical assistance with the immunophenotyping experiments, cell cycle determination and viability assays.
PP2A is a serine/threonine phosphatase that has a critical role in regulating various cellular processes, including protein synthesis, signaling transduction, cell cycle determination, apoptosis, metabolism and stress response.
As our results show, transfection of the most highly UBE2C-expressing C666-1 cells with siRNA for 48 h lead to a 141.6% increase in G2-M and 110.3% increase in S phase, implying a crucial role of UBE2C in NPC cell cycle determination.
Cells were washed three times in PBS 0·1% Triton and resuspended in 300 μl PBS 0·1% Triton + 1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) to counter-stain nuclei for cell cycle determination before analysis by flow cytometry.
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A549-NT cells had a doubling time of approximately 24 hours and A549-T cells of approximately 20 hours as determined by cell cycle determinations (data not shown).
PI staining of methanol-fixed cells was used for cell cycle determinations.
Cell cycle determinations were performed using a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA), and data were interpreted using the CellQuest software, provided by the manufacturer.
Cell cycle distribution determination analysis was performed as previously described [ 19].
The cells were washed, fixed in ethanol for 2 h, and stained with propidium iodide using a Cell Cycle Phase Determination Kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer's protocol.
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