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UV lasers for flow cytometric analysis: HeCd versus argon laser excitation

B Goller and M Kubbies

Department of Cell Biology, Boehringer Mannheim GmbH, Penzberg, Federal Republic of Germany.

Applying flow cytometric single cell analysis, we compared the performance of UV excitation from argon ion and HeCd lasers using various UV-excitable fluorochromes of cell kinetic and cell physiological relevance. The AT-specific DNA fluorochromes DAPI, Hoechst 33258, and Hoechst 33342 showed no significant differences of G1-phase resolution and cell cycle distribution. With the HeCd laser, high-resolution cell kinetic analysis applying the novel BrdU/Hoechst- PI quenching technique showed superior resolution and an almost normalized G2M/G1 channel ratio of the first cell cycle. Indo-1 analysis for detection of intracellular free calcium gave similar results for both excitation sources, although the indo-1 ratio of activated cells was lower for HeCd excitation. Monochlorobimane as an indicator fluorochrome of glutathione content could not be excited sufficiently with the 325-nm line of the HeCd laser and exhibited poor resolution between positive and negative cells. However, the second glutathione-specific fluorochrome o-phtalaldehyde gave even better results with the HeCd laser. Our data indicate that air-cooled HeCd lasers are cheap and reliable UV-excitation sources for most UV- excitable fluorochromes, and might be an alternative to the expensive water-cooled argon and krypton laser.

Volume 40, Issue 4, pp. 451-456, 04/01/1992
Copyright © 1992 by The Histochemical Society


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