Flow cytometric analysis of DNA content of living and fixed cells: a comparative study using various fixatives.
The majority of studies dealing with DNA analyses are made on fixed cells. In this context, the efficiency as fixatives of ethanol, methanol, acetone, Carnoy, Boehm-Sprenger and aldehydes was determined using two different DNA fluorescent probes, Hoechst 33342 and propidium iodide. The purpose of our study was to find the fixative that would provide the best results with respect to the following parameters: aggregates, cell size and granularity, and DNA staining analysis. Using murine fibroblasts, we found that 68% ethanol, 85% methanol and aldehydes did not increase aggregate formation, whereas Carnoy, acetone or Boehm-Sprenger fixatives did. The results show that aldehydes seem to alter cell size least. All fixatives induce an increase in cell granularity, which is very pronounced with alcohols, but aldehydes alter morphology less than alcohols. We observed that the fixatives giving the best resolution with Hoechst 33342 staining lead to a lower measurement variability than with propidium iodide staining. This study leads us to conclude that 68% ethanol and 85% methanol can be considered as appropriate fixatives for flow cytometry studies of DNA content.
Rousselle
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C
Institut Albert Bonniot, Université Joseph Fourier, Dynamique de l'Organisation des Génomes, INSERM U309, La Tronche, France.
Robert-Nicoud
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M
Ronot
X
X
D000818:Animals; D001562:Benzimidazoles; D002449:Cell Aggregation; D002453:Cell Cycle; D002460:Cell Line; D048429:Cell Size; D004247:DNA / Q000032:analysis*; D005347:Fibroblasts; D005404:Fixatives; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes; D051379:Mice; D011419:Propidium