Staining Mouse Bone Marrow Cells with Hoechst to Detect Side Population Cells by Flow Cytometry

To begin, obtain bone marrow cells from the mouse and add five micrograms per milliliter Hoechst 33342 to two milliliters of cell suspension. Prepare the negative control tube as well. Incubate in a water bath at 37 degrees Celsius for 90 minutes with gentle agitation every 30 minutes After incubation, cool the cells on ice for five minutes, and then centrifuge at 250G for five minutes at four degrees Celsius.

Resuspend the cells in a cold running solution containing Hank's Balanced Salt Solution with 2%FBS. Centrifuge again at four degrees Celsius for five minutes. Next, resuspend the resulting cell pellet in 500 microliters of running solution per sample.

Prior to flow cytometry analysis, supplement cells with two micrograms per milliliter of propidium iodide, and place them on ice for approximately five minutes.