Flow cytometry of the Side Population: tips & tricks.

Background: The Side Population (SP) has become an important hallmark for the definition of the stem cell compartment, especially in the detection of these cells and in their physical isolation by fluorescence-activated cell sorting (FACS). SP cells are CD34neg and were discovered using ultraviolet excitation based on the efflux of Hoechst 33342 (Ho342). Although the method works as originally described, we believe that this method is difficult for most investigators.

A rare fraction of human hematopoietic stem cells with large telomeres.

The lack of specific markers for stem cells makes the physical identification of this compartment difficult. Hematopoietic stem cells differ in their repopulating and self-renewal potential. Our study shows that multiple classes of human hematopoietic CD34+ greatly differ in telomere length.

Flow cytometry-based approach to ABCG2 function suggests that the transporter differentially handles the influx and efflux of drugs.

Background: To better characterize the function of the ABCG2 transporter in vitro, we generated three cell lines (MXRA, MXRG, and MXRT) stably expressing ABCG2 after transfection of wild-type ABCG2 and two mutants (R482G and R482T), respectively.

Methods: ABCG2 expression and function were analyzed by flow cytometry using monoclonal antibodies, a variety of fluorescent substrates, and a series of potential inhibitors of the transporter.

Results: ABCG2 expression was detected in all cell lines.