Skip to main content
NCBI home page
Cytotechnology logoLink to Cytotechnology
. 2004 Jul;45(3):155–160. doi: 10.1007/s10616-004-7256-9

High-throughput microtiter assay for Hoechst 33342 dye uptake

Gail M Seigel 1,✉, Lorrie M Campbell 1
PMCID: PMC3450168  PMID: 19003252

Abstract

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.

Keywords: Chemo-resistance, Hoechst 33342 dye, Microtiter assay, Stem cell

Full Text

The Full Text of this article is available as a PDF (202.8 KB).

References

  1. Bhattacharya S., Jackson J.D., Das A.V., Thoreson W.B., Kuszinski C., James J., Joshi S., Ahmad I. Direct identification and enrichment of retinal stem cells/progenitors by Hoechst dye efflux assay. Invest. Ophthalmol. Vis. Sci. 2003;44(6):2764–2773. doi: 10.1167/iovs.02-0899. [DOI] [PubMed] [Google Scholar]
  2. Cone R.D., Grodzicker T., Jaramillo M. A retrovirus expressing the 12S adenoviral E1A gene product can immortalize epithelial cells from a broad range of rat tissues. Mol. Cell. Biol. 1988;8:1036–1044. doi: 10.1128/mcb.8.3.1036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Doyle L.A., Ross D.D. Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2) Oncogene. 2003;22(47):7340–7358. doi: 10.1038/sj.onc.1206938. [DOI] [PubMed] [Google Scholar]
  4. Frankfurt O.S. Increased uptake of vital dye Hoechst 33342 during S phase in synchronized HeLa S3 cells. Cytometry. 1983;4(3):216–221. doi: 10.1002/cyto.990040305. [DOI] [PubMed] [Google Scholar]
  5. Frey T. Nucleic acid dyes for detection of apoptosis in live cells. Cytometry. 1995;21(3):265–274. doi: 10.1002/cyto.990210307. [DOI] [PubMed] [Google Scholar]
  6. Goodell M., Brose K., Paradis G., Conner A.S., Mulligan R.C. Isolation and functional properties of murine hematopoetic stem cells that are replicating in vivo. J. Exp. Med. 1996;183:1797–1806. doi: 10.1084/jem.183.4.1797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kim M.H., Turnquist J., Jackson M., et al. The multidrug resistance transporter ABCG2 (breast cancer resistance protein 1) effluxes Hoechst 33342 and is overexpressed in hematopoietic stem cells. Clin. Cancer Res. 2002;8:22–28. [PubMed] [Google Scholar]
  8. Lahmy S., Viallet P., Salmon J.M. Is reduced accumulation of Hoechst 33342 in multidrug resistant cells related to P-glycoprotein activity? Cytometry. 1995;19(2):126–133. doi: 10.1002/cyto.990190207. [DOI] [PubMed] [Google Scholar]
  9. Maynadie M., Lizard G., Solary E., Roignot P., Carli P.M. Multidrug resistance phenotype evaluation by immunofluorescence and functional tests: comparison of two monoclonal antibodies and three fluorescent dyes in three cells lines. Anticancer Res. 1994;14(6B):2605–2609. [PubMed] [Google Scholar]
  10. Rice G.C., Gray J.W., Dewey W.C. FACS analysis of a hyperthermia-induced alteration in Hoechst 33342 permeability and direct measurement of its relationship to cell survival. J. Cell Physiol. 1985;122(3):387–396. doi: 10.1002/jcp.1041220308. [DOI] [PubMed] [Google Scholar]
  11. Scharenberg CW, Harkey M.A., Torok-Storb B. The ABCG2 transporter is an efficient Hoechst 33342 efflux pump and is preferentially expressed by immature human hematopoietic progenitors. Blood. 2002;99:507–512. doi: 10.1182/blood.V99.2.507. [DOI] [PubMed] [Google Scholar]
  12. Seigel G.M. Establishment of an E1A-immortalized rat retinal cell culture. In Vitro Cell Dev. Biol. 1996;32:66–68. doi: 10.1007/BF02723034. [DOI] [PubMed] [Google Scholar]
  13. Seigel G.M., Sun W., Wang J., Hershberger D., Campbell L., Salvi R. Neuronal gene expression and function in the growth-stimulated R28 retinal precursor cell line. Curr. Eye Res. 2004;28:257–269. doi: 10.1076/ceyr.28.4.257.27831. [DOI] [PubMed] [Google Scholar]
  14. Sery T.W., Lee E.Y., Lee W.H., Bookstein R., Wong V., Shields J.A., Augsburger J.J., Donoso L.A.J. Characteristics of two new retinoblastoma cell lines: WERI-Rb24 and WERI-Rb27. Pediatr. Ophthalmol. Strabismus. 1990;27(4):212–217. doi: 10.3928/0191-3913-19900701-11. [DOI] [PubMed] [Google Scholar]
  15. Summer R., Kotton D.N., Sun X., Ma B., Fitzsimmons K., Fine A. Side population cells and Bcrp1 expression in lung. Am. J. Physiol. Lung Cell. Mol. Physiol. 2003;285:L97–104. doi: 10.1152/ajplung.00009.2003. [DOI] [PubMed] [Google Scholar]
  16. Welm B.E., Tepera S.B., Venezia T., Graubert T.A., Rosen J.M., Goodell M.A. Sca1 positive cells in the mouse mammary gland represent an enriched progenitor cell population. Dev. Biol. 2002;245:42–56. doi: 10.1006/dbio.2002.0625. [DOI] [PubMed] [Google Scholar]
  17. Zhou S., Schuetz J.D., Bunting K.D., Colapietro A.M., Sampath J., Morris J.J., Lagutina J., Grosveld G.C., Osawa M., Nakauchi H., Sorrentino S.P. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat. Med. 2001;7:1028–1034. doi: 10.1038/nm0901-1028. [DOI] [PubMed] [Google Scholar]

Articles from Cytotechnology are provided here courtesy of Springer Science+Business Media B.V.

RESOURCES