Fixation and Permeabilization of Cells for Analysis

For the majority of DNA-binding dyes to be able to quantitatively stain the DNA of cells, they must first be made permeable. This may be achieved either by detergent treatment (e.g., 0.1% Triton X-100) or by fixation. The advantage of fixation is that cells may be kept at 4°C for several days or weeks prior to a

Area Flow Cytometry

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Fig. 7. Doublet discrimination. (A) Signal area and signal height are directly proportional to each other on flow cytometers for single cells and hence when stained with PI, generate a population that forms a 45° line across a dot plot (left plot). G2/M cells are at the top right (center plot), and G0/G1 cells are toward the bottom left of the dot plot (right plot). Cells that fall outside of this relationship are cell aggregates. (B) An alternative is to plot signal area against signal width for a given fluorochrome. This visualizes cell cycle distribution of doublet populations and larger aggregates (as shown).

0 200 400 600 800 1000 Pulse Area

Fig. 7. Doublet discrimination. (A) Signal area and signal height are directly proportional to each other on flow cytometers for single cells and hence when stained with PI, generate a population that forms a 45° line across a dot plot (left plot). G2/M cells are at the top right (center plot), and G0/G1 cells are toward the bottom left of the dot plot (right plot). Cells that fall outside of this relationship are cell aggregates. (B) An alternative is to plot signal area against signal width for a given fluorochrome. This visualizes cell cycle distribution of doublet populations and larger aggregates (as shown).

analysis. There are two principal types of fixatives used: alcohols (generally ethanol or methanol) or aldehydes (generally paraformaldehyde). Alcohol, usually 70% ethanol, is preferred because this is a dehydrating fixative that denatures nuclear proteins and allows better access of the DNA-binding dye to the DNA.

Paraformaldehyde is a crosslinking fixative and, as this locks proteins in position, can lead to suboptimal DNA CVs. However, in some situations (e.g., in which GFP fluorescence needs to be preserved), 1% paraformaldehyde may be used.

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