As stem cells are very important to the organism it is not surprising that they are thought to have very efficient membrane pumps to enable them to protect themselves from influx of potentially harmful chemicals. Certain cell types are known to have hightly efficient pumps for the dyes Rhodamine 123 and Hoechst 33342 in particular.
Hoechst 33342 is a DNA-binding dye that binds preferentially to A-T rich regions of DNA. The dye is excited by UV wavelengths (maximum excitation is 395nm) and emits in the blue (emission maximum is 450nm). However, the emission wavelength shifts to the red end of the spectrum in certain conditions such as apoptosis and also when the dye concentartion is high. This property is also exploited in the identification of putative stem cells in a variety of human and murine cell types. Although Hoechst is able to enter live cells, it is also actively pumped out by ABC (ATP-Binding Cassette) transporters which include p-glycoprotein and ABCG2 in human cells. These transporters may also be specifically inhibited by agents such as verapamil and reserpine.
Margaret Goodell's group was the first to identify a population of cells in bone marrow that appears particularly effective at pumping out the Hoechst dye. These 'side population' or SP cells have been shown to possess stem cell characteristics (a lineage negative phenotype, enriched long-term culture initiating cells). More information may be found at the Goodell Lab's website. Care should be taken when looking for SP cells in other tissues because the identification of this population is dependent on Hoechst concentration and staining time. A kinetic experiment is vital to detemine these parameters.
Below is an example of an SP population in murine bone marrow cells.
It is also possible to combine SP analysis with phenotyping and we have shown that although there is some overlap, not all SP cells have the KTLS phenotype and not all KTLS cells fall in the side population. The cells that show the highest repopualtion potential are those that are SP and KTLS positive.
Data courtesy of Daniel Pearce, Haematopoietic Stem Cell Lab, CRUK.
A detailed protocol for the SP analysis of cells is available here.
