A simple quantitative method of estimation of cell-intactness based on ethidium bromide fluorescence.
A quantitative method based on fluorescence generated by the binding of ethidium bromide (EB) to DNA has been developed for estimation of the intactness of the plasma membrane of a mammalian cell type (goat epididymal spermatozoon). The method consists of mixing of sperm preparations with EB in a modified Ringer's solution followed by immediate measurement of fluorescence intensity at 365-580 nm (excitation-emission). The data were corrected for non-specific values of fluorescence due to intact cells only. The percentage of damaged cells in a sperm population was calculated by comparing the corrected fluorescence values of the cell preparations with those of the sonicated cells. The values of sperm intactness obtained by this method (99.5 +/- 0.3) compared well with those obtained by the widely used "marker enzyme" method (97 +/- 0.8) based on estimation of lactic dehydrogenase in the extracellular medium. The validity of this method has been confirmed by using cells of defined intactness i.e. preparations of vigorously forward-motile spermatozoa that showed nearly 100% intactness. The method can detect as low as 0.5% "leaky" or damaged cells in a cell preparation. The "EB-fluorescence" method is simpler and more rapid and reliable than the conventional "marker enzyme" method for estimation of cellular intactness.
Dey
C S
CS
Indian Institute of Chemical Biology, Jadavpur, Calcutta.
Majumder
G C
GC
D000818:Animals; D015415:Biomarkers; D002470:Cell Survival; D004996:Ethidium; D006041:Goats; D066298:In Vitro Techniques; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D013050:Spectrometry, Fluorescence; D013094:Spermatozoa / Q000166:cytology* / Q000201:enzymology
D015415:Biomarkers; D007770:L-Lactate Dehydrogenase; D004996:Ethidium