The aim of this procedure is to be able to determine the proportion of cells in the different phases of the cell cycle. This is accomplished by first adding the cell proliferation labeling reagent BRDU to a viable cell culture for incorporation into newly synthesized DNA next stain, the BRDU in the cells that have incorporated the reagents, then the DNA is stained with propidium iodide. The final step is to evaluate the proportion of cells in the different phases of the cell cycle by flow cytometry.
Ultimately the cell cycle distribution of a population of experimental cells can be visualized through flow cytometric analysis of BRDU labeled cells. The main advantage of this technique over existing methods like tri to thyme incorporation and scintillation counting is that with BRD labeling, the proportion of cells in each cell cycle phase can be determined. Visual demonstration of this message is critical as the fellow cytometric analysis set are difficult to explain without accompanying images of the different parameters that need to be adjusted.
Demonstrating the procedure will be two graduate students from my laboratory, Matt Chii and me Miri. ADD BRDU at a one to 1000 dilution to the experimental cell cultures incubate for one hour to harvest the cells, aspirate the culture medium, and then wash the cells thoroughly with PBS two times to remove all traces of medium. This time using PBS supplemented with three millimolar EDTA, rinse the cultures quickly once more, and then aspirate the P-B-S-E-D-T-A solution thoroughly to detach the cells.
Add around 0.5 milliliters of P-B-S-E-D-T-A to each six centimeter culture dish and incubate the dishes at 22 degrees Celsius for approximately five minutes. Then transfer the cell cultures from each plate to individual 15 milliliter conical tubes. Now centrifuge the cells at 500 times gravity for five minutes at room temperature and then remove the supinate, then resuspend the cells in 100 microliters of PBS.
Finally, fix the cells by adding five milliliters of 95%ethanol dropwise while vortexing centrifusion the cells at 500 times gravity for five minutes of room temperature. Once again, remove the ethanol and re suspend the pellet in one milliliter of two normal HCL containing 0.5%Triton X 100 by adding the solution to the cells in a dropwise fashion while vortexing then incubate the cells at room temperature for 30 minutes. After pelleting the cells again aspirate the S supernatant taking special care as the cells form a very loose pellet at this step.
Then gently re suspend the pellet in one milliliter of 0.1 molar sodium tetra bore rate and incubate the cells again. Now pellet the cells once more and this time incubate them in 0.5 milliliters of antibody solution containing mouse anti BRDU antibodies diluted one to 50 after pelleting the cells again reus. Suspend this pellet in 50 microliters of antibody solution containing rabbit anti-US ZI conjugated secondary antibodies, diluted one to 25.
Then incubate the cells in the dark for 30 minutes of room temperature. After centrifuging the cells of final time, incubate them in 0.5 milliliters of propidium iodide and RNA solution in the dark at 37 degrees Celsius for 30 minutes. Finally, pass the digested cell solution through a cell strainer to remove aggregates.
Then transfer the single cells to an appropriate tube for sample uptake on the flow cytometer for asynchronously proliferating cell populations such as the one shown. In this example, place a gate around the two most abundant peaks in the doublet discriminating plot to select single cell events for all subsequent analysis. Then analyze an asynchronously proliferating culture sample to serve as a positive control for staining and flow cytometer parameter set up.
Adjust the sensitivity of the photomultiplier tube for propidium iodide staining such that the two N and four N peaks from the single cells are centered at the arbitrary units of 200 and 400 on the x axis. Next, adjust the sensitivity of the FXI detecting photo multiplier tube such that FXI negative cells are above background levels. BRDU positive cells should be approximately 10 times brighter and the x axis should be displayed as a logarithmic scale.
The single events that are captured by the flow cytometer form a horseshoe shaped arc from the G one phase in the lower left up to the S phase and then back down to the lower right for the G two M phase. As seen in this representative figure, collect 5, 000 to 10, 000 single cell events to achieve the desired range of DNA content. After measuring propidium iodide and BRDU content, assign the cells to the G one s or G two MPH phase with gates around the two BRDU negative populations centered at 200 for G one and 400 for G two M.Include all the events above these two boxes in a single gate for measuring the S-phase, the percentage of cells in each gate will represent the relative number of cells in the G one s and G two M phases as demonstrated here in this bar graph for the previous scatterplot figure.
After staining for a cell marker of interest in this example CD 20 centrifuge the cells at 500 times gravity for 10 minutes at 22 degrees Celsius, and then resuspend the pellet in five milliliters of PBS supplemented with 1%BSA. After pelleting the cells again at 500 times gravity for five minutes of room temperature use 0.5 milliliters of the propidium iodide and RNA solution to rehydrate the cells. Then stain and digest the cells as just shown now gate the two n and four end single cell peaks, and then adjust the sensitivity of the photomultiplier tube for FXI detection such that the unstained cells are above background levels.
Ideally, the CD 20 FXI positive cells will be 10 to 100 times more intensely stained than the background. Again, the x axis should be displayed as a logarithmic scale scale. Finally, select the CD 20 positive cells that are 10 times brighter than background levels and with propidium iodide staining between 200 and 400 for display in a propidium iodide versus cell counts histogram as shown here in this sample figure, collect at least 1000 CD 20 positive events for each sample to limit variability in this figure.
The three-dimensional flow cytometric analysis of propidium iodide and BRDU stain cells is displayed. Note that the cells with two n and four NDNA content are centered over the 200 and 400 marks on the X axi scale. For propidium iodide staining intensity, BRDU staining intensity is measured on a logarithmic scale.
On the Y axis, note the positions of gates used to quantitate cells in the G one s and G two M phases of the cell cycle. The relative proportion of cells in each of the G one s and G two M gates from the previous figure are shown here. Here, propidium, iodide and CD 20.
Staining from mixture of cells, some of which ectopically expressed CD 20 and PRB is shown. Note that the cells with two n and four NDNA the most abundant populations are centered over the 200 and 400 marks on the x axis. The position of the CD 20 positive gait select cells that are stained at least 10 times more brightly than background.
This histogram represents cell counts versus propidium iodide staining for CD 20 negative cells from previous scatterplot that are asynchronously proliferating in this histogram CD 20 positive cells from the earlier CD 20 scatterplot that have been induced to arrest with p rrb expression and contain cells with primarily two NDNA content are shown. These data demonstrate that an arrested subpopulation can be distinguished from other cells in the culture using this staining technique. In the next three figures, data from flow cytometric analysis of retroviral expression of the Cyclin-dependent kinase inhibitor P 27 KIP one in mouse embryonic fibroblasts are shown here.
Propidium iodide, BRDU analysis was used to measure the cell cycle phases in an asynchronously proliferating population of cells that were transduced with an empty P babe retroviral vector as shown here. Few BRDU positive events were evident in the S-phase gate in response to P 27. Note also the greater intensity of events in the G one and G two M gates.
Likewise, the percentage of cells in S-phase for P 27 expressing cells is quite low as diagrammed in this figure. This type of analysis has been very effective in characterizing cell cycle control deficits in cells derived from various strains of gene targeted mice. In these next two figures, unformed memory epithelial cells were treated with the growth inhibitory cytokine TGF beta one for 24 hours.
As shown in this first figure, BRDU labeling in S-phase cells is greatly diminished by TGF beta one signaling. Note the accumulation of cells primarily in the G one phase of the cell cycle as can be seen in the middle panel. Quantification of the different phases of the cell cycle confirms that TGF beta one primarily inhibits proliferation in the G one phase of the cell cycle leading to an accumulation in this phase.
In this last series of experimental analysis, PRB deficient, SAS two cells were transfected with CMV CD 20 expression vector and either CMV RB or CMV beta GAL as a control. This first figure demonstrates the cell cycle distribution of the negative control transfected cells compared to their distribution following 72 hours of PRB expression. Note, the almost exclusive presence of a two N peak in the right hand panel in this final figure curve fitting by multi-cycle software revealed that the cells accumulated primarily in the G one phase following PRB expression.
While the populations in the s and G two M phases were relatively depleted. Once mastered, this technique can be adapted to study other cell cycle markers such as phosphorylate histones in mph. After watching this video, you should have a good understanding of how to determine the cell cycle distribution of a population of cells.
Don't forget the two normal hydrochloric acid is caustic and propidium. Iodide is a potential carcinogen. They can be hazardous in.
Precautions should always be taken while using these chemicals.
