Stem Cell Identification by Side Population Analysis

The original protocol developed by Margaret Goodell’s laboratory measures the PI emission through the same filter used for the Ho342 red. However, based on the emission properties of this dye, and for a more precise discrimination of dead cells, an alternative procedure was chosen in my laboratory. Propidium Iodide (PI) emission is measured on a logarithmic scale using a different PMT (BP 630/30) and 488-nm excitation, instead of using the same PMT for both PI and Ho342 red emission, resulting in better resolution of the SP. In my opinion, a feasible live gate based on use of these conditions should better resolve the SP, even if apoptotic/prenecrotic cells are present in the sample.

All human samples should be stained using the same incubation times (2 hr). Specimens obtained from other species may require different incubation times, which should be determined in pilot experiments. Murine samples should be stained for 90 min. After red-cell lysis, cells are resuspended at a density of 1-2 × 10E6 cells/ml in DMEM supplemented with 2% heat-inactivated fetal bovine serum (FBS) and 10 mM HEPES, prewarmed to 37º C. Hoechst 33342 (Ho342) is added at a concentration of 5 or 10 μg/ml, depending on the density of cells per ml. Cells are incubated 2 hr (human samples) or 90 min (murine samples) in a water bath at 37º C with periodic agitation. Cells are then centrifuged 6 min at 483 × g, 4º C, and resuspended at a concentration of 1-2 × 10E7 cells/ml in cold HBSS containing 2% FBS and 10 mM HEPES. Samples should be kept at 4º C until analysis. PI is added at a concentration of 5 μg/ml to exclude dead cells. In order to remove cellular aggregates, cells should be filtered through a 50-μm nylon mesh prior to analysis.

For immunofluorescence assays, directly conjugated antibodies are preferred. However, indirect immunofluorescence techniques can also be applied for polychromatic flow cytometry. Staining with directly conjugated antibodies allows a feasible multicolor analysis, even using high-power laser settings. If low antigen expression is expected, use monoclonal antibodies conjugated to bright fluorochromes (e.g., phycoerythrin-conjugated anti-CD34 antibody). Conversely, and for the highest expected antigen expression, use monoclonal antibodies conjugated to less bright fluorochromes (e.g., fluorescein-conjugated CD45 antibody). If needed for high-power lasers, a set of neutral-density filters can be used to avoid saturation of the photomultiplier tubes. Conversely, in order to detect low-fluorescence signals, neutral-density filters can be removed if they are being used.

More recently, the cell-permeant nucleic acid-binding Vybrant® DyeCycle™ Violet stain (DCV), which has emission characteristics similar to those of Hoechst 33342, but with a longer wavelength excitation maxima (369 nm), has been also validated to study and visualize DNA in living cells as well as to allow side population analysis on flow cytometers with violet lasers. Using DCV and violet excitation, the SP displays almost identical distributions as for the Ho342 SP assay.

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Goodell, M.A., Rosenzweig, M., Kim, H., Marks, D.F., DeMaria, M., Paradis, G., Grupp, S.A., Sieff, C.A., Mulligan, R.C., and Johnson, R.P. 1997. Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat. Med. 3:1337-45.

Zhou, S., Schuetz, J.D., Bunting, K.D., Colapietro, A.M., Sampath, J., Morris, J.J., Lagutina, I., Grosveld, G.C., Osawa, M., Nakauchi, H., and Sorrentino, B.P. 2001. 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. 7:1028-1034.

Goodell, M.A. Stem cell identification and sorting using the Hoechst 33342 Side Population. Current Protocols in Cytometry. Chapter 9: Unit 9.18, 2005.

Petriz, J. Flow cytometry of the Side Population (SP). Current Protocols in Cytometry. Unit 9.23, 2007.

Telford W.G., Bradford J., Godfrey W., Robey R.W., Bates S.E. 2007. Side population analysis using a violet-excited cell-permeable DNA binding dye. Stem Cells 25(4):1029-36.