FACS analysis of a hyperthermia‐induced alteration in Hoechst 33342 permeability and direct measurement of its relationship to cell survival

Rice, Glenn C.; Gray, Joe W.; Dewey, William C.

doi:10.1002/jcp.1041220308

Cited by 11 publications

(2 citation statements)

References 24 publications

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“…Dye uptake measurements on a per-well and per-cell basis may depend upon a number of factors, including cell size, adherence vs. suspension culture, the phase of the cell cycle and metabolic activity. Uptake of Hoechst 33342 is increased during S phase in HeLa cells (Frankfurt 1983), as well as in cells exposed to hyperthermia (Rice et al 1985). These factors need to be taken into account for well-by-well comparisons of different cell types.…”

Section: Discussionmentioning

confidence: 99%

High-throughput microtiter assay for Hoechst 33342 dye uptake

Seigel¹,

Campbell²

2004

Cytotechnology

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Exclusion of Hoechst 33342 dye is a characteristic common to stem cells, as well as chemotherapy-resistant cancer cells. Normally, these dye-excluding cells can be sorted from enzymatically dissociated tissues with a UV cell sorter/flow cytometer. UV-flow cytometry can be expensive, time-consuming and not readily available to all laboratories. We have developed a simple, high-throughput 96-well microtiter plate assay by which cell populations can be quickly screened for Hoechst dye uptake and exclusion. The method is compatible with green-fluorescent EGFP expressing cells, often used in stem cell biology. Useful applications for this assay will be the rapid screening of clonal stem cell populations and tumor cells for Hoechst dye uptake.

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Section: Discussionmentioning

confidence: 99%

High-throughput microtiter assay for Hoechst 33342 dye uptake

Seigel¹,

Campbell²

2004

Cytotechnology

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“…It has not been the scope of this report, and it had not been possible either, to get a complete knowledge of all membrane damaging and -interfering agents, but merely to outline differences between the various types of membrane damage. In addition to the types of membrane damage tested here a few more could be mentioned: mechanical damage; hyperthermia that may partly act as a membrane damage [17], and freezing of cells. Most other types of membrane damage may be classified in one of the above groups as for instance complement-or virus-in duced membrane damage that may be ex plained more or less by a detergent-like ef fect [18].…”

Section: Discussionmentioning

confidence: 99%

Importance of the Cellular Energy Level for Enzyme Release Induced by Direct Membrane Damage

Kristensen

1990

Enzyme

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A release of intracellular enzymes may occur as a result of energy depletion of the cells or after direct membrane damage. A direct membrane damage, however, may be counteracted by the cell by energy-consuming reactions, thus more or less being dependent on the cellular energy level. Therefore, the association between enzyme release and the energy level was investigated after addition of various agents impairing the normal membrane function, i.e. lysophosphatidylcholine, phospholipases, the Ca ionophore A23187, ouabain and superoxide/H(2)O(2), and after incubation in a hypotonic medium. It was observed that in some types of membrane damage the cellular energy is minimally involved, in other types the extent of enzyme release depends on the cellular energy level, and in some other types the cellular energy is affected but the connection to the enzyme release is not clear. The results also indicate that the effect of membrane-active agents arising in ATP-depleted states may be more severe in ATP-depleted than in normal cells.

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Modulation of adriamycin transport by hyperthermia as measured by fluorescence-activated cell sorting

Rice

Hahn

1987

Cancer Chemother. Pharmacol.

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Heat-induced (45.5 degrees C) modification of adriamycin uptake and efflux were measured by flow cytometry in CHO cells in vitro. Administration of adriamycin with simultaneous 15-min or 30-min heat treatment increased drug uptake in a dose-dependent manner. Fluorescence-activated cell sorting showed that cytotoxicity to adriamycin was correlated with relative cellular concentration (fluorescence) for both unheated cells and those heated and simultaneously treated with adriamycin. However, if adriamycin administration followed the heat treatment, accumulation was significantly reduced, primarily as a result of decreased passive drug diffusion (rather than increased efflux) in the heated cells. Cells made heat-tolerant by prior heating also exhibited reduced adriamycin uptake 12 h later, and further heating did not increase uptake. Cell sorting experiments indicated that cytotoxicity of adriamycin was not necessarily correlated with intracellular drug levels when drug administration followed the heat treatment. Also, heat-sterilized cells exhibited a two-fold increase in adriamycin uptake over surviving cells, as assessed by simultaneous measurement of dansyl lysine and adriamycin content. These results indicate that sensitization to adriamycin by simultaneous heat treatment is probably due to increased drug uptake. The decreased sensitization observed when drug administration is followed by heating is probably the result of both decreased uptake and decreased drug DNA accessibility.

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FACS analysis of a hyperthermia‐induced alteration in Hoechst 33342 permeability and direct measurement of its relationship to cell survival

Cited by 11 publications

References 24 publications

High-throughput microtiter assay for Hoechst 33342 dye uptake

High-throughput microtiter assay for Hoechst 33342 dye uptake

Importance of the Cellular Energy Level for Enzyme Release Induced by Direct Membrane Damage

Modulation of adriamycin transport by hyperthermia as measured by fluorescence-activated cell sorting

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