DNA Content and Apoptosis
Note: In order to use soluble Fas ligand or anti-Fas mAb to induce apoptosis the cells of interest must have the Fas receptor on the cell surface.
Cells are prone to aggregation with ethanol fixation so expect some loss. Cell fixation should be optimized for each cell type by increasing fixation time and/or ethanol concentration, and PI concentration. Samples in 70% Ethanol can be stored at -200C for weeks. For a PDF formatted printable copy of the protocol click on the DNA Content Protocol above.
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, or when a silicon sampling tube is employed. 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 and contain a 2C DNA content and can be used as an external standard. Chicken red blood cells (CRBC) are 35% of normal G1 (2C) and areplaced at a mean channel of 70. When used as an internal standard the 2C G1 peak will present itself at channel 200. The CRBC iscommercially available. A poorly prepared single cell suspension may present false peaks that the analytical software will interpret as an aneuploid population.
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 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
Last updated: 11-March-02
Copyright © 1996-2003. Department of Carcinogenesis, The University of Texas M.D. Anderson Cancer Center, Smithville, Texas.
All rights reserved.