Chapter 9 Supravital Cell Staining with Hoechst 33342 and DiOC5(3)

Crissman, Harry A.; Hofland, M.H.; Stevenson, Anita P.; Wilder, Mark E.; Tobey, Robert A.

doi:10.1016/s0091-679x(08)60514-2

Cited by 19 publications

(12 citation statements)

References 11 publications

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“…The Thrl4 and Tyrl5 kinase activities were also examined in HeLa cells that were delayed in their progression through G2 by the DNA binding dye, Hoechst 33342. Hoechst 33342 binds to DNA, and interferes with the proofreading mechanisms, which occur during G2, thus delaying the initiation of mitosis (Crissman et al, 1990). When Hoechst 33342 is added to cells following synchronization by a double thymidine block, activation of histone Hi kinase activity is delayed by >4 h relative to control cells, indicative of a delay in entry into mitosis (Figure 4).…”

Section: Discussionmentioning

confidence: 99%

“…Nocodazole arrested cells were obtained by incubating cells in the presence of 0.1 Mg/ml nocodazole (Sigma) for 10 to 18 h. Mitotic cells were then collected by mitotic shake off. Cells were delayed in G2 by the addition of 7.5 ug/ml Hoechst 33342 (Calbiochem, San Diego, CA) 4 h after release from a double thymidine block (Crissman et al, 1990). Plates were frozen at -80°C until needed.…”

Section: Hela Cell Culture and Synchronizationmentioning

confidence: 99%

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Cell cycle regulation of the p34cdc2 inhibitory kinases.

Atherton-Fessler

Liu

Gabrielli

et al. 1994

MBoC

113

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In cells of higher eukaryotic organisms the activity of the p34cdc2/cyclin B complex is inhibited by phosphorylation of p34cdc2 at two sites within its amino-terminus (threonine 14 and tyrosine 15). In this study, the cell cycle regulation of the kinases responsible for phosphorylating p34cdc2 on Thr14 and Tyr15 was examined in extracts prepared from both HeLa cells and Xenopus eggs. Both Thr14- and Tyr15- specific kinase activities were regulated in a cell cycle-dependent manner. The kinase activities were high throughout interphase and diminished coincident with entry of cells into mitosis. In HeLa cells delayed in G2 by the DNA-binding dye Hoechst 33342, Thr14- and Tyr15-specific kinase activities remained high, suggesting that a decrease in Thr14- and Tyr15- kinase activities may be required for entry of cells into mitosis. Similar cell cycle regulation was observed for the Thr14/Tyr15 kinase(s) in Xenopus egg extracts. These results indicate that activation of CDC2 and entry of cells into mitosis is not triggered solely by activation of the Cdc25 phosphatase but by the balance between Thr14/Tyr15 kinase and phosphatase activities. Finally, we have detected two activities capable of phosphorylating p34cdc2 on Thr14 and/or Tyr15 in interphase extracts prepared from Xenopus eggs. An activity capable of phosphorylating Tyr15 remained soluble after ultracentrifugation of interphase extracts whereas a second activity capable of phosphorylating both Thr14 and Tyr15 pelleted. The pelleted fraction contained activities that were detergent extractable and that phosphorylated p34cdc2 on both Thr14 and Tyr15. The Thr14- and Tyr15-specific kinase activities co-purified through three successive chromatographic steps indicating the presence of a dual-specificity protein kinase capable of acting on p34cdc2.

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Section: Discussionmentioning

confidence: 99%

Section: Hela Cell Culture and Synchronizationmentioning

confidence: 99%

Cell cycle regulation of the p34cdc2 inhibitory kinases.

Atherton-Fessler

Liu

Gabrielli

et al. 1994

MBoC

113

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“…Unfortunately, the choice of such fluorochromes is limited. Hoechst 33342 is one of such dyes and when used in combination with the membrane potential sensing dye DiOC5(3) offers relatively good resolution in measurement DNA content of live cells 8. The dye is excited at UV wavelength (350 nm) and fluoresces in blue (460 nm).…”

Section: Supravital Cell Stainingmentioning

confidence: 99%

Analysis of Cellular DNA Content by Flow and Laser Scanning Cytometry

Darzynkiewicz¹,

Halicka²,

Zhao³

2010

Advances in Experimental Medicine and Biology

147

124

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This chapter covers several aspects of methodology of DNA content analysis in individual cells that is most commonly used for assessment of DNA ploidy and for enumeration of cells in particular phases of the cell cycle. Briefly presented are general principles of instrumentation and cell analysis by flow- and laser scanning- cytometry. Described are major methods designed to stain DNA with fluorochromes in live cells, in detergent-permeabilized cells, in cells fixed prior to DNA staining as well as in nuclei of cells isolated from paraffin-embedded tissues. Briefly addressed are approaches to estimate cellular DNA content in conjunction with cellular immunophenotype. Discussed are factors that affect accuracy of DNA content measurement such as: (i) differences in chromatin structure of the analyzed cells that restrict DNA accessibility to fluorochromes, (ii) stoichiometry of interaction between fluorochromes and DNA in chromatin and (iii) chemical mass action law defining dependency of fluorochrome binding to DNA in relation to fluorochrome concentration and number of potential binding sites in a sample. Described also are controls used to ensure accuracy of DNA ploidy determination, the principles in ploidy assessment and possible pitfalls in analysis.

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“…Separation of the cells according to the phase of the cell cycle was performed using a supravital staining with a DNA binding dye (Hoechst 33342) in combination with the membrane potential modifying fluorochrome DiOC 5 [22]. During the last hour of culture, T-I cells were incubated with Hoechst 33342 (5 g/ml) and DiOC 5 (0.3 g/ml).…”

Section: Separation Of Cells According To the Phase Of The Cell Cyclementioning

confidence: 99%

Proliferation and Differentiation of Rat Theca-Interstitial Cells: Comparison of Effects Induced by Platelet-Derived Growth Factor and Insulin-Like Growth Factor-I1

Dulęba

Spaczyński²,

Arıcı

et al. 1999

Biology of Reproduction

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This study was designed to evaluate mechanisms regulating proliferation of steroidogenically active and steroidogenically inactive theca-interstitial (T-I) cells, and, specifically, to evaluate the effects of platelet-derived growth factor (PDGF) and insulin-like growth factor-I (IGF-I). T-I cells obtained from immature Sprague-Dawley rats were cultured in chemically defined media. Proliferation was assayed by thymidine incorporation and cell counting. Steroidogenically active cells were identified by the presence of 3beta-hydroxysteroid dehydrogenase activity. Flow cytometry facilitated separation of dividing cells (in S and G2/M phases of the cell cycle) from nondividing cells (in G0 and G1 phases of the cell cycle). PDGF alone (0.1-1 nM) produced a dose-dependent increase in DNA synthesis by up to 136%. IGF-I alone (10 nM) increased DNA synthesis by 56%. In the presence of both IGF-I (10 nM) and PDGF (0.1-1 nM), DNA synthesis increased by 108-214%. PDGF (1 nM) increased the total number of T-I cells by 43%; this effect was due to an increase in the number of steroidogenically inactive cells (47%). In contrast, the stimulatory effect of IGF-I (10 nM) was predominantly due to an increase in the number of steroidogenically active cells (163%). Separation of dividing cells from nondividing cells was accomplished with the aid of flow cytometry. In the absence of growth factors, the proportion of steroidogenically active cells was 35% lower among proliferating than resting cells. PDGF (1 nM) decreased the proportion of steroidogenically active cells among both proliferating and resting cells (by 43% and 16%, respectively). In contrast, IGF-I (10 nM) increased the proportion of steroidogenically active cells among proliferating cells by 56%. These findings indicate that differentiated/steroidogenically active cells divide; furthermore, PDGF and IGF-I may selectively stimulate proliferation of individual subpopulations of T-I cells, thereby providing a mechanism for development of structural and steroidogenically active components of the T-I compartment.

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Chapter 9 Supravital Cell Staining with Hoechst 33342 and DiOC5(3)

Cited by 19 publications

References 11 publications

Cell cycle regulation of the p34cdc2 inhibitory kinases.

Cell cycle regulation of the p34cdc2 inhibitory kinases.

Analysis of Cellular DNA Content by Flow and Laser Scanning Cytometry

Proliferation and Differentiation of Rat Theca-Interstitial Cells: Comparison of Effects Induced by Platelet-Derived Growth Factor and Insulin-Like Growth Factor-I1

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