Advanced Cell Cycle Analysis Lab
The laboratory will be centered on multi-parametric cell cycle analysis of human endothelial cells.  The lab will be in three parts.  There is no “wet-lab” component.
The first part is flow cytometry of replicating cells that have been stained for DNA content, cyclins A2 and B1, and phospho-S10-histone H3. The objective will be to “generate” data that will support an analysis that reduces the cell cycle to the phases: G1, S, G2, and M, and further subdivides M into 6 states (PR1-3, PR4, PM, M, LM1, and LM2).  States are defined by the correlated levels of expression of the employed markers. This part of the lab will be led by Mike Sramkoski who will discuss cell preparation and staining while demonstrating data acquisition on a BD Biosciences LSR II.
The second part is laser scanning cytometry (LSC) of cells stained in the same manner as above. The difference is that the endothelial cells have not been trypsinized and are fixed in the state that we are interested in probing. The objective will be to “generate” data that support a similar analysis to that done on the flow cytometry data, but here we will correlate the mitotic states with cell morphology and thus, mitotic states, using the imaging capability of the Compucyte, iCyte. Tammy Stefan will lead this part of the lab and discuss cell preparation and staining while demonstrating data acquisition on the iCyte.
The third part of the lab will be analytical and will be led by Jim (Jake) Jacobberger. Mike and Tammy will have already demonstrated primary gating to preprocess data (compensation; aggregate and debris removal) and secondary gating to obtain frequencies of phases and states on the two platforms using FACS Diva and iGeneration software. Dr. Jacobberger will demonstrate the use of WinList (Verity Software House) to obtain expression profiles (cell cycle related expression of a biomarker) and use of that knowledge to use Gemstone to obtain the same phase/state information without gates.