Reference
Ormerod, M.G., Ed.: Flow Cytometry A Practical Approach, Second Ed. 1992, Oxford University Press, Inc., New York, NY
Notes:
Preparing a single cell suspension*, counting cells, determining viability and recovery as well as estimating cell doubling time are critical factors which provide information for precise and accurate interpretation of the cell cycle. Cell cycle phases and distributions are affected by chemical treatment where cell accumulation or loss within a given cell cycle compartment may be due to programmed cell death entry, changes in the cell's doubling time, or interuption of a biological process such as DNA synthesis or mitosis.
Cells are prone to aggregation with ethanol fixation so expect some loss. Any enzymatic detachment (trypsin, collagenase, dispase, etc.) must be executed with care to avoid damage to the cell membrane and agglutination of proteins or DNA leakage resulting in excessive aggregates. Cell fixation should be optimized for each cell type by increasing or decreasing the fixation time and/or ethanol concentration (70% - 90%). PI concentration can range from 40ug/ml - 60ug/ml. For optional fixation protocols and other staining procedures that might be more applicable to a particular cell type visit our references page. Samples in 70% Ethanol can be stored at -200C for several days. For a PDF formatted printable copy of the protocol click on the DNA Content Protocol .
Often the question arises, "How few cells can I submit for analysis?" Cell density plays an important role in efficient and accurate acquisition for DNA content with an intercalating dye. Our standard acquisition is 20,000 single events which doesn't sound like much, but when staining is not homogenous and/or aggregates are present as many 500,000 may have to be acquired or lost until a stable G1 peak can be obtained. Propidium iodide, an intercalating molecule binds stoichiometrically and is not base specific. If the DNA content is too great the PI is not kept at saturation and there is a loss of consistant staining. This is apparent when factors such as too great a cell density, aneuploidy, a G2 arrestment, endoreduplication, incomplete fixation, or when a silicon sampling tube is employed. A poorly prepared single cell suspension, incomplete fixation, or non-homogenous staining may present false peaks or wide CV's that the analytical software will interpret as an aneuploid population.
For large or elongated cells such as astrocytes, glials, epithelials, fibroblasts, keritinocytes, etc. acqusition times can be from 6-10 minutes per sample.Typically 500,000 cells/ml in the above protocol will provide a good analysis. Cell counting is important not only for staining but to ensure a representative population. By performing a cell count at the harvest and later when resuspending in propidium iodide one may determine how much cell loss occured and whether this will affect the experimental results.
For ploidy analysis and instrument standardization the following controls may be considered. Normal mouse thymocytes are >98% G1, containing a 2N DNA content and can be used as an external standard. Chicken red blood cells (CRBC) are 35% of normal G1 (2N) and are placed at a mean channel of 70. When used as an internal standard the 2N G1 diploid peak will present itself at channel 200. The CRBC is commercially available. The nuclei of the plant Nicotiana tabacum may be used as an external standard since it has a prominent G1 peak with a 2N DNA content.
Researchers must possess some knowledge about their cells, i.e. lineage, mutations, karotype, etc. An additional parameter or second color can provide greater information. The thymidine analog bromodeoxyuridine is employed for absolute S-phase and Cyclin B1 identifies G2/M arrest, aneuploidy or endoreduplicating cells. See references.
Cell Cycle Kinetics
Accurate cell cycle kinetics requires rigorous quantitation of the three phases of the cell cycle as well as the rate of DNA synthesis. In the late 1950's cell cycle kinetics was limited to the determination of a cell's doubling time (easy) and mitotic index (challenging). H3-thymidine (pulse) first accurately determined cell proliferation (DNA synthesis) by assuming that either a cell incorporates H3-thymidine or it was lost and any residual H3-thymidine was not sequestered in other cellular organelles. With the advent of flow cytometry DNA Content became the next vogue in cell cycle kinetics because it could accurately quantitate the three phases of the cell cycle and was relatively easy as well as inexpensive in terms of cost and time. However, should the cell be undergoing extensive DNA repair in it's S-phase, stop synthesizing ("S0") DNA, or fail a S-phase checkpoint that initiates programmed cell death (apoptosis) there will be a discrepancy in the correlation between the number of cells in the S-phase (DNA Content) and the cell's proliferation (H3-thymidine - doubling time). This is due to the fact that cells in S-phase that stop synthesizing DNA will label positive for S-phase with propidium iodide (PI) while not incorporating any H3-thymidine. For example many lymphocyte lineage cell lines show an increase in their S-phase when given reagents to induce apoptosis since they have a S-phase entry pathway or checkpoint for programmed cell death. In the 1980's with introduction of a highly specific monoclonal antibody against an incorporated thymidine analog, bromodeoxyuridine (BrdU) Investigators could now combine DNA Content with a second color for accurate cell proliferation. This absolute-S application in addition to quantitating DNA synthesis allows significant resolution of early S-phase from late G1 as well as late S-phase from early G2 making elaborate and expensive software programs using algorithms unnecessary. Cell cycle transition times vary with cell type, growth conditions, etc. Typical phase transition times are approximately12 hours for G1, 6 hours for S, 4 hours for G2, and 0.5 hours for M.
Cell Cycle Analysis
This is only a broad guideline to determine confidence in the cell preparation and represents the facility manager's opinion only and does not reflect the software manufacturer's (MultiCycle, Phoenix Flow Systems, Inc., San Diego, CA) opinion.
G1 CV < 8.0
G2/G1 ~ 1.97 (1.8 - 1.9 acceptable)
% B.D. < 15 (background/debris/aggregates)
Debris < 30
Chi Sq < 10 Estimate of how far from the model the data deviates
To learn more about cell cycle modeling and analysis of DNA Content:
Shackney, S.E. and Shankey, T.V. 1999 Cell Cycle Models for Molecular Biology and Molecular Oncology: Exploring New Dimensions Cytometry 35:2 p. 97-116
Shankey, T.V., Rabinovitch, P.S., Bagwell, B., Bauer, K.D., Duque, R.E., Hedley, D.W., Mayall, B.H., and Wheeless, L. 1993 Guidelines for Implementation of Clinical DNA Cytometry Cytometry 14: p.472-477
All the below examples employed aggregate discrimination to provide 20,000 single cells for analysis by MultiCycle cell cycle analytical software distributed by Phoenix Flow Systems, Inc.
Astrocytes
In the above example a mouse astrocyte cell line (immortalized with SV40) is labeled with PI according to the protocol at the top of the page. The cells have a good Chi Sq., are stained homogenously with low CV's and little debris. However due to cell aggregates a significant population is observed in the 4N cell position as well as a pronounced 8N population. Typical aggregate discrimination techniques failed to separate single cells from aggregates. Standard cell cycle analysis interprets these aggregates as a second cycling cell population with a G1 at channel ~195 and G2 at channel ~382. Upon cell counting employing a hemocytometer and stage microscope the following cell populations were identified. Approximately 450,000 single cells/ml; 130,000 doublets/ml; 30,000 triplets/ml; 30,000 quadruplicates/ml; and 20,000 quintuplet cells/ml. While single cells present a symmetrical orientation with respect to the laser beam, cell aggregates pose asymmetrical problems as well as DNA content shifts rendering the data unreliable. The above profile is typical of aneuploid cells. If it is determined with microscopy that a single cell suspension produced the above profile the cells should be submitted to a cytogenetics lab for karotyping. The Molecular Cytogentics Facility is part of M.D. Anderson's CCSG Shared Resources. Another explanation for the above profile is endoreduplication, where the diploid cells have failed mitosis and enter the G1 phase with a 4N DNA content.
Osteosarcoma
In the above example a human osteogenic sarcoma cell line (Saos-2) is labeled with 7-AAD according to the protocol at the top of the page. Though the Chi Sq. is good and the cells have a resolvable sub-G1 peak they are not stained homogeneously exhibiting large CV's. These cells require optimization of the fixation/permeabilization and staining protocol. A literature search for publications relevant to this type of cell may provide a more suitable protocol for this application. After careful optimization of the labeling protocol the Saos-2 cells showed significant improvement in resolving their DNA profile as shown below.
Breast Cancer
In the above example a human breast adenocarcinoma cell line (MCF-7) is labeled with PI according to the protocol at the top of the page. This cell line is not as easily labeled as mouse thymocytes but as this example shows excellent data may be achieved with optimization of the staining protocol. In the example below MCF-7 cells (from the same experiment) are stimulated to a higher level of proliferation as indicated by a significant increase in the S-phase cell population. The homogeneity of staining and CV's remain consistent.
Dead Cell Discrimination
Propidium Iodide (or 7-AAD) may be used in cell surface labeling (or immunophenotyping) to exclude dead cells from the analysis. Cells should not be fixed/permeabilized prior to running on the flow cytometer. Cells are incubated on ice for 1 - 5 minutes in PI at a final concentration of 2ug/ml. The below profile shows how the live cells exclude the PI while those cells that have compromised cytoplasmic membranes fluoresce from uptake of the dye.
Cell Surface Labeling 3-Color Intracellular BrdU and Cell Surface 2-Color Cell Cycle Cell Surface and DNA Content
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Last updated: 06-November-06
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