|Side population protocol
G. V. Denis laboratory 2007
Isolation of population enriched for hematopoietic stem cells from mice.
1. All steps must be performed under sterile conditions. Obtain 3 donor mice and isolate femurs, tibias and sternums under sterile conditions, dunking tissues in 70% ethanol and using sterile dissecting forceps, tweezers and scalpels.
2. Remove skin and hair.
3. Carefully remove as much muscle, fat and connective tissue as possible from bones. Steps 1 – 3 should take about 1 hour.
4. Load a 4 ml Dounce homogenizer with the bones and cover with Hanks buffered salt solution. Break bones and homogenize to release bone marrow cells into suspension.
5. Filter suspension through 70 m mesh to remove bone and tissue fragments.
6. Centrifuge at 250 x g into pellet and aspirate away supernatant.
7. Resuspend pellet in 3 ml RBC lysis buffer (eBioscience) at 37 degrees and keep in incubator for three minutes.
8. Add 10 ml serum-containing medium, mix, centrifuge to pellet.
9. Resuspend in media again, filter out clumps with 70 m mesh and count density of cell population.
10. Adjust cell suspension to 1 x 106/ml with pre-warmed media and add sterile Hoechst 33342 to 5 g/ml.
11. Keep in incubator for 90 minutes exactly.
12. Remove, place on ice; spin down supernatant in pre-chilled centrifuge at 4 degrees; recover pellet and resuspend in sterile phosphate buffered saline containing 2 % serum and 2 g/ml propidium iodide. Filter for MoFlo separation; recover cells into medium containing serum.
13. Isolate “side population” of Hoechst-excluding cells by red vs blue fluorescence; also exclude PI positive cells. Yield of cells should be between 0.1 and 0.25% of unfractionated BM. Recoveries from 3 donor mice range from 40,000 to 100,000 stem cells. Mo Flo separation requires 90 minutes to 2 hours.
14. Cells should be allowed to recover in incubator by efflux of remaining Hoechst dye, which is quite toxic.
The “side population” is transitory, not stable, and its appearance and visualization by flow cytometry is a function of the concentration of Hoechst, the time of incubation, the quality of the cells, and other important factors like temperature and pH. Each investigator should optimize the time required for the population to appear. Too long an incubation in dye or too high a concentration will cause the population to vanish.
Incubation of the Hoechst staining cells with verapamil (50 M) from the beginning of the exposure to the dye will block the transporter that effluxes the dye, and all cells will become Hoechst positive, or the transitory population will occur early and will not persist. The disappearance of the “side population” from flow cytometry plots confirms that these are the cells that harbor the verapamil-sensitive transporter. We have independently verified that the side population stains positive for stem cell markers such as Sca1.