Flow Cytometry Laboratory

Cell Cycle Analysis



The measurement of the DNA content of cells was one of the first major applications of flow cytometry and is still one of the most common applications today. The DNA content of the cell can provide a great deal of information about the cell cycle, and consequently the effect on the cell cycle of added stimuli eg transfected genes or drug treatment. We can also combine the measurement of DNA content with the quantitation of an antigen, so that we can assess its expression during the cell cycle or alternatively look at the DNA profile of a subset of cells defined by this antigen.

Single colour DNA Staining

Bivariate staining with Bromodeoxyuridine

DNA plus antigen staining








TopSingle colour DNA staining


The DNA of cells can be stained by a number of dyes. Examples include:

The most commonly used DNA dye is propidium iodide (PI), which intercalates in the DNA helix and fluoresces strongly red (emission maximum 637nm). It has the advantage that it is excited by 488nm light and can be used on most common benchtop flow cytometers. However it does require cells to be fixed or permeabilised and therefore non-viable. PI also stains double-stranded RNA and this should be removed with ribonuclease.

An alternative is to employ Hoechst 33342 which binds to AT-rich regions in the DNA and will enter viable cells without the need for fixation, so cells can be recovered and grown afterwards. The rate of dye uptake is dependent on dye concentration and cell type. The disadvantage with this method is that Hoechst 33342 is UV-excited so cannot be used on most benchtop flow cytometers. However it may allow dye combinations that are not possible with propidium iodide.

These two dyes are the standards used here at ICRF.

The protocol for DNA staining by propidium iodide is as follows:

After harvesting and washing cells, fix in ice-cold 70% ethanol (ethanol in distilled water) while vortexing. Samples can stay in ethanol for up to 7 days at 4°C.

Spin at 2000rpm, 5 mins. Wash x2 in PBS.

Treat cells with 100l of100g/ml ribonuclease for 5 minutes at room temperature.

Add 400l propidium iodide (50g/ml). Analyse by flow cytometry using 488nm excitation, gating out doublets and clumps using pulse processing and collecting fluorescence above 620nm. A typical profile looks like this.



The protocol for DNA staining by Hoechst 33342 is as follows:

Treat the cells with Hoechst 33342 for 10-60 minutes at 37°C. The concentration of Hoechst used must be pre-determined, as the optimal concentration will vary with cell type, but will probably be in the range 5-20g/ml.

Spin at 1000rpm, 5 mins. Wash x2 in PBS. If appropriate add propidium iodide to identify dead cells.

Analyse by flow cytometry, using ultra-violet and 488nm excitation, collect emitted Hoechst fluorescence between 390nm and 480nm.


PULSE PROCESSING

Most DNA profiles can be improved if cell doublets are identified and excluded from analysis. This is done by analysing the pulse produced as a particle passes through the laser beam. Two G1 cells stuck together will appear to have the same amount of DNA as a single G2 cell. However, since they are bigger they will take slightly longer to pass through the laser beam, so by analysing pulse width versus pulse height or area we can eliminate the majority of doublets from the anlaysis.
This example shows the effect of removing cell doublets from the analysis



TopBivariate Staining

The use of the bromodeoxyuridine (BrdU) method for the analysis of the cell cycle has proved very useful in the study of cell kinetics. Bromodeoxyuridine is an analogue of the DNA base thymidine and competes with that base for uptake during the synthesis of DNA. So only those cells that have been actively synthesising DNA during the time that BrdU is present will be positive for it. It can be detected by a monoclonal antibody and by simultaneously staining for DNA content with propidium iodide, the percentage of cells in G1, S and G2/M can be determined. By altering the time that BrdU is present cell cycle times can also be assessed. The protocol used in this Lab is as follows:


Treat cells with 10M BrdU for an appropriate time (30mins - several days)

After harvesting and washing cells, fix in ice-cold 70% ethanol (ethanol in distilled water) while vortexing. Samples can stay in ethanol for up to 7 days at 4°C.

Spin at 2000rpm, 5 mins. Wash x2 in PBS.

Treat cells with 2M Hydrochloric acid for 20 mins at room temperature with frequent mixing.

Spin off acid (2000rpm, 5 mins), wash x2 in PBS and x1 in PBS-T (PBS + 0.5% Tween + 0.05%w/v BSA).

Add 2l anti-BrdU antibody (Becton Dickinson) directly to the cell pellet, leave for 15 mins at room temperature. Wash x2 in PBS-T. It is best to titrate each antibody batch to determine optimal dilution.

Add 50l FITC-conjugated rabbit anti-mouse immunoglobulins (1:10, DAKO), leave for 15 mins at room temperature. Wash x1 in PBS-T.

Add 100l of 100g/ml Ribonuclease, and 400l propidium iodide (50g/ml). Analyse by flow cytometry ,using pulse processing to gate out cell doublets and clumps.

This profile shows the distribution obtained after a 60 minute pulse of BrdU.



TopDNA plus antigen staining



Optimal fixation for DNA staining by propidium iodide is 70% ethanol, although this may alter some antigens even if they are stained prior to fixation. Paraformaldehyde (0.5-2%) is the fixative of choice for optimal antigen staining preservation because of its cross-linking action but may lead to sub-optimal DNA profiles.

Permeabilisation with, for example, saponin, will also enable internal antigen staining and will give acceptable DNA profiles. Therefore some preliminary experiments may be required to determine the best method. We have found that many antigens may be stained with FITC-labelled antibodies and then fixed in 70% ethanol. Analysis then enables the percentage of cells expressing the antigen to be determined and the DNA profiles of positive and negative cells to be analysed.

This example is of CD2-transfected cells.

DNA plus antigen staining is also possible using Hoechst 33342, although this does require a dual laser set-up. However, it does have the added advantage that, depending on the fluorochromes used for antigen detection, dead cells may also be excluded from the analysis. Using this method, the cell cycle status of phenotypically-defined sub-populations can be assessed.




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