UV lasers for flow cytometric analysis: HeCd versus argon laser excitation.
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.
Department of Cell Biology, Boehringer Mannheim GmbH, Penzberg, Federal Republic of Germany.
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B
Goller
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M
Kubbies
D001128:Argon; D006690:Bisbenzimidazole; D001973:Bromodeoxyuridine; D002104:Cadmium; D002118:Calcium / Q000378:metabolism*; D002455:Cell Division; D002460:Cell Line; D004247:DNA / Q000032:analysis*; D005434:Flow Cytometry / Q000379:methods*; D005978:Glutathione / Q000032:analysis; D006371:Helium; D006801:Humans; D007211:Indoles; D007834:Lasers
D007211:Indoles; D002104:Cadmium; D006371:Helium; C007293:DAPI; D001128:Argon; D004247:DNA; D001973:Bromodeoxyuridine; D005978:Glutathione; D006690:Bisbenzimidazole; C048960:indo-1; D002118:Calcium