Hoechst fluorescence intensity can be used to separate viable bromodeoxyuridine-labeled cells from viable non-bromodeoxyuridine-labeled cells.
BACKGROUND
5-Bromo-2'-deoxyuridine (BrdU) is a powerful compound to study the mitotic activity of a cell. Most techniques that identify BrdU-labeled cells require conditions that kill the cells. However, the fluorescence intensity of the membrane-permeable Hoechst dyes is reduced by the incorporation of BrdU into DNA, allowing the separation of viable BrdU positive (BrdU+) cells from viable BrdU negative (BrdU-) cells.


METHODS
Cultures of proliferating cells were supplemented with BrdU for 48 h and other cultures of proliferating cells were maintained without BrdU. Mixtures of viable BrdU+ and viable BrdU- cells from the two proliferating cultures were stained with Hoechst 33342. The viable BrdU+ and BrdU- cells were sorted into different fractions from a mixture of BrdU+ and BrdU- cells based on Hoechst fluorescence intensity and the ability to exclude the vital dye, propidium iodide. Subsequently, samples from the original mixture, the sorted BrdU+ cell population, and the sorted BrdU- cell population were immunostained using an anti-BrdU monoclonal antibody and evaluated using flow cytometry.


RESULTS
Two mixtures consisting of approximately 55% and 69% BrdU+ cells were sorted into fractions consisting of greater than 93% BrdU+ cells and 92% BrdU- cells. The separated cell populations were maintained in vitro after sorting to demonstrate their viability.


CONCLUSIONS
Hoechst fluorescence intensity in combination with cell sorting is an effective tool to separate viable BrdU+ from viable BrdU- cells for further study. The separated cell populations were maintained in vitro after sorting to demonstrate their viability.
Mozdziak
P E
PE
Department of Poultry Science, North Carolina State University, Raleigh, North Carolina 27695, USA. pemozdzi@unity.ncsu.edu
Pulvermacher
P M
PM
Schultz
E
E
Schell
K
K
D016475:3T3 Cells; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000963:Antimetabolites / Q000494:pharmacology; D001562:Benzimidazoles / Q000494:pharmacology*; D001973:Bromodeoxyuridine / Q000276:immunology / Q000494:pharmacology*; D002469:Cell Separation; D002470:Cell Survival; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000494:pharmacology; D007202:Indicators and Reagents / Q000494:pharmacology; D007364:Intercalating Agents / Q000494:pharmacology; D008297:Male; D051379:Mice; D008938:Mitosis; D018482:Muscle, Skeletal / Q000166:cytology; D011419:Propidium / Q000494:pharmacology; D051381:Rats
D000911:Antibodies, Monoclonal; D000963:Antimetabolites; D001562:Benzimidazoles; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D007364:Intercalating Agents; D011419:Propidium; D004247:DNA; D001973:Bromodeoxyuridine; C017807:bisbenzimide ethoxide trihydrochloride