a heterogeneous and multifaceted entity, comprising cells that are partly biased towardmyeloid or lymphoid phenotypes. Identifying the rare cell population, on which thehematopoietic homeostasis is elegantly built, represents therefore one of the major
challenges in the field (2–4). Nonetheless, despite the numerous efforts, a single specificmarker, that can be employed alone to isolate HSCs, has yet to be discovered. Hence,investigators must turn to combinations of different markers or physiological properties.Benefiting from the advances in multicolor flow cytometry and monoclonal antibodydevelopment, several laboratories have proposed over the last two decades differentisolation schemes that, however, lead to extremely similar HSC populations (5, 6).
Among the principal criteria utilized for HSC identification and isolation is the expression,or lack of expression, of specific cell surface markers. The isolation of one of the mostthoroughly characterized populations of HSCs relies on the positive expression of thetyrosine kinase receptor c-Kit (CD117) and the membrane glycoprotein Sca-1 (7),
concomitantly with the lack of markers of terminal differentiation (Ter119, Gr-1, Mac-1,B220, CD4, and CD8), collectively known as Lineage markers. The resulting c-Kit+ Sca-1+Lineage- population, commonly referred to as KSL cells, contains cells capable ofhematopoietic reconstitution. However, different studies showed that the KSL fraction
contains a variety of progenitors, including ST-HSCs. Thanks to the contribution of differentgroups, schemes to further enrich the KSL fraction in HSCs have been developed over time.These strategies are based on either the combination with other surface markers, such asThy1.1 (KSL Thylow or KTSL), CD34 (KSL CD34neg/low), and Flk2 (KSL CD34− Flk2−)(8), or on physiological properties, such as the capability to efflux Hoechst observed in SPcells (SPKSL or SPKLS, pronounced SParKLeS) (4, 7, 9, 10).
More recently, alternative methods to identify HSCs have been described, that do not rely onthe KSL scheme. These strategies include the use of markers such as Tie-2 (11), Endoglin(12), or endothelial protein C receptor (EPCR) (13). Morrison and colleagues recently
described an alternative method based on markers from the signaling lymphocytic activationmolecule (SLAM) family (CD150+ CD244− CD48−) (14). However, in order to obtain highpurity, this strategy should be used in conjunction with other purification schemes.In this chapter, we will focus on the purification of murine SPKLS cells, based on thepeculiar pattern that bone marrow cells acquire after Hoechst 33342 staining.
Hoechst 33342 fluorescent dye is a bisbenzimidazole derivative, capable of permeating
through cell membranes and binding to nucleic acids. The emission of fluorescence is highlyaffected by DNA properties, such as chromatine rearrangements, DNA conformation, andnucleic acid composition. In particular, Hoechst dyes bind in a stoichiometric manner to AT-rich regions of the minor groove of double-stranded DNA (this property has beenextensively used by genetists to develop the Q-bands staining for chromosomes).
Interestingly, when Hoechst dyes bind to DNA, their fluorescence undergoes a smallspectral shift, that can be detected and used as a measurement of the amount of cellular
DNA. This property has been exploited in flow cytometry to study ploidy and distribution inthe different cell-cycle stages of a heterogeneous population, such as bone marrow samples.Traditionally, cell cycle studies have been performed by analyzing Hoechst emission at ashort wavelength (450 nm), through a “blue” bandpass on a fluorescence-activated cell-sorter. However, Hoechst fluorescence can be detected with “red” (650 nm) bandpass opticsas well. When Hoechst blue and red fluorescence signals are simultaneously collected andplotted against each other, a characteristic tail-shaped population, displaying low
fluorescence, can be observed and distinguished from the main bulk that conversely emitshigh levels of fluorescence. This “tail” is the so-called Side Population, or SP, and
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Rossi et al.Page 3
comprises cells that display low Hoechst fluorescence. Conversely to the main bulk of bonemarrow cells (whose Hoechst fluorescence is directly proportional to the DNA content), theatypical cytometric morphology of SP cells is a direct consequence of their capability toefflux with high efficiency the vital dye Hoechst 33342. However, what makes this peculiarbone marrow population so interesting for the stem cell field is the fact that SP cells arehighly enriched in HSCs, capable of sustaining multilineage and long-term engraftment inthe murine model. Since the first description of SP cells in 1996 (10), follow-up studies alsoproved that the SP fraction encompasses entirely the hematopoietic activity that resides inthe murine bone marrow, thus making Hoechst staining a unique experimental tool in stemcell biology (2, 4–6, 15).
The capability of SP cells to efflux vital dyes at a higher rate than other bone marrow cells isbelieved to reside in the activity of membrane pumps belonging to the superfamily of ATP-binding cassette (ABC) transporters. Members of this family are, for instance, multidrugresistance 1 (murine Mdr 1a/1b; human MDR1) and breast cancer resistance protein 1(Bcrp1)/ABC, superfamily G, member2 (ABCG2). Interestingly, drugs such as verapamilblock the activity of these transporters and concomitantly cause the SP profile to disappear.Knock-out and retroviral-driven overexpression models helped shed some light onto the roleABC transporters play in HSC biology. MDR1 overexpression only slightly increases the SPfraction; on the other hand, Mdr 1a/1b−/− bone marrow shows numbers of SP cells
comparable to the wild type, thus indicating that this membrane transporter only plays amarginal role in the SP phenotype (16, 17). Conversely, the enforced expression of ABCG2significantly expands SP cells, while loss of ABCG2 expression has been shown to
drastically reduce the size of the SP fraction. Nonetheless, since HSC numbers and functionin these mice are preserved, it is not yet clear whether the efflux plays a functional role inHSCs. Furthermore, ABCG2 knock-out mice still contain in their bone marrow a few
residual SP cells, suggesting that multiple drug transporters are likely to be involved in theappearance of this phenotype (18–21).
However, if ABC membrane pumps are not crucial determinants of stem cell activity, whyare they expressed at high levels in stem cells? This observation could be teleologicallyinterpreted as a mechanism that biological systems adopt to protect from the environmentcrucial subsets of cells, like HSCs. Also, membrane pumps could play a role in extrudingdifferentiation factors from HSCs, thus helping maintaining their stemness throughout thelife of an organism.
NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript2. Materials
2.1. Sample Preparation: Isolation of Murine Bone Marrow Cells
1.2.
Murine bone marrow cells obtained from C57Bl/6 mice, 5–8 weeks old (see Note1).
HBSS. Hanks Balanced Salt Solution, supplemented with 2% Fetal Bovine Serumand 10 mM HEPES buffer. The solution so prepared will be hereafter referred to asHBSS+.
Needles (27 Gauge and 18 Gauge).
3.
1 This protocol was originally established and optimized for murine bone marrow cells, derived from normal C57Bl/6 mice. Becauseof the high sensitivity of Hoechst efflux to multiple parameters, we strongly recommend investigators, who are attempting thisprocedure for the first time, to follow the protocol exactly as we describe, until proficiency in SP staining and identification is
achieved. In order to optimize the protocol for different species, we suggest to change one parameter at a time (for instance, durationof the staining or Hoechst concentration).
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Rossi et al.Page 4
4.5.
Cell strainer (70 μm).
Red Blood Cells (RBC) lysis buffer. 0.17 M TrisCl, pH 7.6:0.16 M NH4Cl = 1: 9.
NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript2.2. Staining of Murine Bone Marrow Cells with Hoechst 33342
1.
DMEM. Dulbecco’s Modified Eagle’s Medium with High Glucose, supplementedwith 2% Fetal Bovine Serum and 10 mM HEPES buffer. The solution so preparedwill be referred to as DMEM+.
Hoechst 33342, bisBenzimide H33342 trihydrochloride (Sigma-Aldrich). To makeconcentrated stock solutions of Hoechst 33342, dissolve the powder in water(recommended concentration: 1 mg/mL, 200× solution) and filter-sterilize (seeNote 2).
Verapamil (Sigma-Aldrich). Prepare a concentrated stock (100×) in 95% Ethanoland use at the final concentration of 50 μM in the staining buffer (HBSS+ andHoechst 33342) (see Note 3).
Circulating water bath at exactly 37°C (see Note 4).Refrigerated centrifuge at 4°C (see Note 5).
2.
3.
4.5.
2.3. Isolation of SP Sca-1+ c-Kit+ Lineage− Cells
1.2.3.4.5.6.
HBSS+ (as described in Subheading 2.1).
Anti-Sca-1 antibodies either biotinylated or FITC-conjugated (BD Pharmingen).Anti-Biotin magnetic microbeads (Miltenyi Biotech).AutoMACS separator (Miltenyi Biotech).
Anti-c-Kit antibody. We use a PE-conjugated antibody.
Anti-Lineage antibody cocktail. The cocktail comprises a mixture of the followingPE-Cy5-conjugated antibodies (all from eBioscience): anti-B220, anti-CD4, anti-CD8, anti-Gr-1, anti-Mac-1, and anti-TER119.
Propidium Iodide (PI, Sigma-Aldrich). Prepare a stock solution at 10 mg/mL inwater and store at −20°C. From this solution, prepare a working solution at 200 μg/mL and keep it at 4°C, protected from light. The final concentration of PI in thesample should be 2 μg/mL (100× dilution of the working solution).
7.
2.4. Identification and Sorting of SPKLS Cells
1.
Flow cytometer equipped with a UV laser, such as a MoFlo sorter (Dako) or aFACSAria (BD Biosciences) (see Note 6).
2For long-term storage, prepare aliquots of the stock solution (e.g., 1 mL aliquots) and store them at −20°C, protected from light.Avoid, when possible, repeated thawing/freezing cycles. We strongly recommend using a new Hoechst aliquot for each experiment.3Verapamil is a drug that blocks the activity of the membrane transporters responsible for the efflux of Hoechst 33342. When
Verapamil (50 μM) is included in the Hoechst staining solution and in the washing buffers, the SP fraction is no longer detectable andbecomes part of the main population. We highly recommend the use of Verapamil-treated cells as negative control to help
investigators identify the “true” SP population and draw the sorting gate. However, once the method has been routinely established,Verapamil treatment can be left out.
4Hoechst staining is highly sensitive to temperature. Therefore, the water bath must be set at precisely 37°C. Avoid using water bathswhose temperature fluctuates (we recommend using a circulating water bath) and avoid immersing ice-cold or frozen reagents into thewater during the staining.
5Use a refrigerated centrifuge for spinning cells down and always keep the sample at 4°C or on ice. In the case the stained cells areexposed to higher temperatures, they might expel Hoechst to the point they will become undistinguishable from the “true” SP cells.This will eventually affect the composition and decrease the purity of the SP.
6Although it is possible to detect SP using a violet laser, in order to obtain optimal results, we recommend using a UV laser.
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Rossi et al.Page 5
3. Methods
Because the method relies on detecting dye efflux from a cell, which is a dynamic biologicalprocess, a successful SP staining is highly dependent on cell and Hoechst concentration, aswell as temperature and time of staining. Even small variations in any of these parameterscan affect significantly the composition and purity of the SP. Here we illustrate the protocolas it was originally established for the staining of C57Bl/6 bone marrow and we recommendthe protocol to be followed exactly as we describe before attempting the use in differentspecies, tissues, or mouse strains.
3.1. Sample Preparation: Isolation of Murine Bone Marrow Cells
1.2.3.
Anesthetize the mouse and sacrifice it by cervical dislocation. Lay the mouse on itsback and profusely spray with 70% Ethanol to sterilize.
Make a horizontal abdominal incision at the level of the knees and pull the skinuntil the legs are exposed completely.
Proceed to remove the tibias by cutting through the ankles and the knees. Clean themuscle off the tibias and place them in a Petri dish containing HBSS+ (5 mL) onice.
Proceed now to remove the femurs, by cutting at the level of the hips. Carefullyremove the muscle from the femurs and put them into the Petri dish with the tibias.Femurs are extremely rich in bone marrow, so we recommend to cut off the bone asclose to the hip as possible.
Load a 10cc syringe with HBSS+ buffer and, holding a bone over a new Petri dish,insert the needle (27 Gauge) into one of the extremities and proceed to flush thebone marrow out of the bone. As the bone marrow is expelled, the bones will
appear clearer. Repeat the same by inserting the needle into the second extremity ofthe same bone and flush thoroughly (see Note 7).
Using a syringe with an 18-Gauge needle, proceed to resuspend the bone marrow inthe Petri dish. Repeat several times (four to five times), until the clusters of bonemarrow will convert into a homogeneous single-cell suspension. Pay special
attention to avoid the formation of air bubbles while resuspending cells, because oftheir detrimental effect on cell survival. Transfer the cell suspension into a 50 mL-conical tube and filter through a 70 μm cell-strainer to remove from the sample cellclumps or bone fragments.
Carefully count the bone marrow cells, paying particular attention to exclude redblood cells (RBCs) (see Note 8). To do so, prepare a 1:20 dilution of an aliquot ofbone marrow cell suspension (e.g., 5 μL) in RBC-lysis buffer (95 μL) for counting.One C57Bl/6 mouse (5–8 weeks old) will averagely yield 5–7 × 107 nucleatedcells. Note that, in order to proceed to the following staining, no Ficoll separationor lysis of red blood cells of the whole sample is necessary.
NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript4.
5.
6.
7.
3.2. Staining of Murine Bone Marrow Cells with Hoechst 33342
1.
Pre-warm the staining medium (DMEM+) in a circulating water bath at 37°C.
7When isolating tibias and femurs, it is important to remove as much muscle as possible in order to prevent the bone marrow fromsticking to it once it is flushed out of the bone.
8Cell dilution, Hoechst concentration, and staining time are all critical factors in determining an optimal staining. In particular, dyeconcentration and number of nucleated cells should be carefully determined.
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2.3.4.
Spin down bone marrow cells and resuspend in pre-warmed DMEM+ at theconcentration of 106 cells/mL (see Notes 8 and 9).
Add Hoechst 33342 to the cell suspension to a final concentration of 5 μg/mL(from the 200× working solution).
Incubate the sample for exactly 90 min at 37°C in a circulating water bath. Duringthe incubation, periodically mix the tubes and always ensure that the tubes are fullyimmersed in the water.
Once the 90-min staining is completed, always keep your sample at 4°C and alwaysuse a refrigerated centrifuge to spin cells down, in order to prevent continuousHoechst expulsion from the stained cells (see Note 10).
Spin down the Hoechst-stained cells in a refrigerated centrifuge and resuspend iniced HBSS+ buffer at the concentration of 108 cells/mL. Bone marrow cells arenow stained with Hoechst and ready for the following staining procedures withmonoclonal antibodies. Any further handling of the sample must be performed at4°C or on ice (see Note 11).
NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript5.
6.
3.3. Isolation of SP Sca-1+ c-Kit+ Lineage− Cells
1.
Sca-1 enrichment (see Note 12). Incubate cells on ice in the presence of anti-Sca-1biotinylated antibody (0.5 μg/106 cells, 1:100 dilution) (see Note 13). After 10 min,wash out the unbound antibody by adding a tenfold volume of iced HBSS+. Spincells down at 4°C and resuspend in HBSS+ buffer.
Label bone marrow cells with anti-biotin magnetic micro-beads (1:5 dilution).Incubate for 15 min at 4°C.
Wash the sample with a tenfold volume of HBSS+ buffer and spin cells down at4°C.
Resuspend at 2 × 108 cells/mL in HBSS+. The sample is now ready to be processedby AutoMACS (choose the program for stringent positive selections) (see Note 14).Spin down at 4°C the Sca-1-enriched cells and resuspend in iced HBSS+ buffer.Label the cells with anti-c-Kit antibody and with an anti- Lineage cocktail,comprising anti-B220, anti-CD4, anti-CD8, anti-Gr-1, anti-Mac-1, and anti-TER119 antibodies. Although the sample has been previously enriched for Sca-1+cells, we recommend staining the sample with an anti-Sca-1 antibody as well, as acontrol during the sorting. Incubate for 15 min on ice.
2.3.4.5.6.
9In order to prevent cells from sticking to the plastic, we recommend using polypropylene tubes while staining with Hoechst.
10Because of the aforementioned sensitivity of the procedure to temperature, even when the staining process is over, the samples mustbe maintained at 4°C, in order to prohibit efflux of the dye from the cells. Therefore, whether you are going to directly sort SP cells oryou are going to perform antibody staining, always keep your sample at 4°C.
11If interested in SP isolation only, disregard the following KSL staining. Resuspend the sample in HBSS+ buffer and PI and proceedto sort. However, keep in mind that combination of SP staining with KSL markers significantly increases HSC purity, other than beingan internal diagnostic parameter for optimal staining conditions. Likewise, if this protocol is used to isolate stem cells from othertissues, SP staining should be combined, whenever possible, with tissue-specific stem cell markers.
12Enrichment of the bone marrow sample before sorting is not strictly necessary, but strongly recommended. Enrichment helpsincrease purity and yield after sorting and sensibly decreases sort time.
13The antibody concentration of 0.5 μg/106 cells reflects the optimal staining conditions that have been identified in our laboratoryand is consistently used for each antibody mentioned throughout this protocol. However, especially for samples different from murinebone marrow cells, we recommend to adjust the antibody titration ad hoc.
14Alternatively, the Sca-1 enrichment can be performed manually using Miltenyi MS/LS columns for positive selection.
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Rossi et al.Page 7
7.
Wash the sample with a tenfold volume of HBSS+ buffer and spin cells down at4°C. Resuspend HBSS+ buffer containing PI. The sample is now ready for sortingof SP c-Kit+ Lin− Sca-1+ cells.
NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript3.4. Identification and Sorting of SPKLS Cells
1.
Excitation of Hoechst 33342. In order to view the SP, the flow cytometer must beequipped with a high power ultraviolet laser (35–100 mW), which is capable toexcite both Hoechst 33342 and Propidium Iodide (PI) at 350 nm (see Note 15). Asecond laser is necessary to excite additional fluorochromes involved in thestaining, such as a 488 nm laser for FITC and Phycoerthrin.
Detection of Hoechst 33342 emission. The emission of Hoechst 33342 is measuredbimodally and commonly referred to as Hoechst Blue and Hoechst Red. HoechstBlue is measured with a 450BP filter, whereas Hoechst Red is measured with a675LP filter. In order to separate the different emission wavelength, a dichroicmirror is used (we use a 610 DMSP). PI emission is also measured with the 675LPfilter, but its signal is significantly brighter than the one captured for Hoechst Red,so that PI-positive cells line up to the very far right side of the SP profile (Fig. 1).FACS Analysis. The characteristic SP profile can be visualized by plotting HoechstBlue emission (on the vertical axis) vs. Hoechst Red emission (on the horizontalaxis). The detectors for both parameters must be set on linear mode. The voltagemust be adjusted so that the PI-positive dead cells will appear at the far rightvertical line. Also, if the voltage is set correctly, red blood cells should grouptogether in the lower left corner. The majority of the bone marrow cells will bedisplayed in the central area or in the upper right quadrant of the plot. If the
cytometer settings are arranged correctly, the SP profile should appear as displayedin Fig. 1.
Identification and gating of SPKLS cells. Once the instrument set-up has been
performed, follow the gating strategy described in Fig. 2. Briefly, start by drawingthe first gate around the SP population. Proceed by checking the morphologicalphenotype of SP cells (FSC vs. SSC plot) and gate out all the events not compatiblewith stem cell morphology (low granulosity and small/medium size). Finally,proceed to analyze the KSL phenotype: first, gate Lineage− cells and then displaythese events as shown in the last panel of Fig. 2. The events that simultaneouslyfulfill the criteria of both c-Kit and Sca-1 positivity represent the desired SPKLSpopulation (see Note 16).
2.
3.
4.
References
1. Dykstra B, Kent D, et al. Long-term propagation of hematopoietic differentiation programs in vivo.Cell Stem Cells. 2007; 1:218–29.
2. Camargo FD, Chambers SM, et al. Hematopoietic stem cells do not engraft with absoluteefficiencies. Blood. 2006; 107:501–7. [PubMed: 16204316]
15In the case that the sorting strategy relies also on conjugated antibodies (as in the case of SPKLS purification), the flow cytometermust have the corresponding additional lasers (e.g., a 488 nm laser, if cells are stained with FITC and PE).
16Despite the unique pattern of SP cells, uninitiated investigators usually are challenged by deciding where to draw the SP gate,especially when it comes to deciding how far toward the top of the tail it is possible to go, without including cells that are not “true”HSCs. In our laboratory, we tend to use a conservative gate, while attempting to maximize cell yield and minimize contaminationfrom non-HSCs. An excellent internal quality control for drawing the SP gate in the correct position is provided by the KSL stainingitself. Since SP cells are highly enriched in HSCs, the SP gate should not contain more than 25% Lineage+ cells. Also, approximately85% of SP should be KSL. If these criteria are not matched, it generally means that a more restricted gate should be drawn. Anotherpossible reason is that the protocol has been poorly performed and consequently a high percentage of non-SP cells are contaminatingthe SP gate.
Methods Mol Biol. Author manuscript; available in PMC 2013 April 10.
Rossi et al.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptPage 8
3. Morrison SJ, Weissman IL. The long-term repopulating subset of hematopoietic stem cells isdeterministic and isolatable by phenotype. Immunity. 1994; 1:661–73. [PubMed: 7541305]4. Goodell MA, Rosenzweig M, et al. Dye efflux studies suggest that hematopoietic stem cellsexpressing low or undetectable levels of CD34 antigen exist in multiple species. Nat Med. 1997;3:1337–45. [PubMed: 9396603]
5. Weksberg DC, Chambers SM, et al. CD150-side population cells represent a functionally distinctpopulation of long-term hematopoietic stem cells. Blood. 2008; 111:2444–51. [PubMed: 18055867]6. Pearce DJ, Ridler CM, et al. Multiparameter analysis of murine bone marrow side population cells.Blood. 2004; 103:2541–6. [PubMed: 144998]
7. Okada S, Nakauchi H, et al. In vivo and in vitro stem cell function of c-kit-and Sca-1-positivemurine hematopoietic cells. Blood. 1992; 80:3044–50. [PubMed: 1281687]
8. Christensen JL, Weissman IL. Flk-2 is a marker in hematopoietic stem cell differentiation: a simplemethod to isolate long-term stem cells. Proc Natl Acad Sci USA. 2001; 98:14541–6. [PubMed:11724967]
9. Challen GA, Boles N, et al. Mouse hematopoietic stem cell identification and analysis. CytometryA. 2009; 75:14–24. [PubMed: 190231]
10. Goodell MA, Brose K, et al. Isolation and functional properties of murine hematopoietic stem cells
that are replicating in vivo. J Exp Med. 1996; 183:1797–806. [PubMed: 8666936]
11. Arai F, Hirao A, et al. Tie2/angio-poietin-1 signaling regulates hematopoietic stem cell quiescence
in the bone marrow niche. Cell. 2004; 118:149–61. [PubMed: 15260986]
12. Chen CZ, Li M, et al. Identification of endoglin as a functional marker that defines long-term
repopulating hematopoietic stem cells. Proc Natl Acad Sci USA. 2002; 99:15468–73. [PubMed:124386]
13. Balazs AB, Fabian AJ, et al. Endothelial protein C receptor (CD201) explicitly identifies
hematopoietic stem cells in murine bone marrow. Blood. 2006; 107:2317–21. [PubMed:16304059]
14. Kiel MJ, Yilmaz OH, et al. SLAM family receptors distinguish hematopoietic stem and progenitor
cells and reveal endothelial niches for stem cells. Cell. 2005; 121:1109–21. [PubMed: 159959]15. Challen GA, Little MH. A side order of stem cells: the SP phenotype. Stem Cells. 2006; 24:3–12.
[PubMed: 149630]
16. Bunting KD, Galipeau J, et al. Transduction of murine bone marrow cells with an MDR1 vector
enables ex vivo stem cell expansion, but these expanded grafts cause a myeloproliferativesyndrome in transplanted mice. Blood. 1998; 92:2269–79. [PubMed: 97467]
17. Bunting KD, Galipeau J, et al. Effects of retroviral-mediated MDR1 expression on hematopoietic
stem cell self-renewal and differentiation in culture. Ann N Y Acad Sci. 1999; 872:125–40.discussion 140–1. [PubMed: 10372117]
18. Bunting KD, Zhou S, et al. Enforced P-glycoprotein pump function in murine bone marrow cells
results in expansion of side population stem cells in vitro and repopulating cells in vivo. Blood.2000; 96:902–9. [PubMed: 10910903]
19. Scharenberg CW, Harkey MA, et al. The ABCG2 transporter is an efficient Hoechst 33342 efflux
pump and is preferentially expressed by immature human hematopoietic progenitors. Blood. 2002;99:507–12. [PubMed: 11781231]
20. Zhou S, Morris JJ, et al. Bcrp1 gene expression is required for normal numbers of side population
stem cells in mice, and confers relative protection to mitoxantrone in hematopoietic cells in vivo.Proc Natl Acad Sci USA. 2002; 99:12339–44. [PubMed: 12218177]
21. Zhou S, Schuetz JD, et al. 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. 2001;7:1028–34. [PubMed: 11533706]
Methods Mol Biol. Author manuscript; available in PMC 2013 April 10.
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Fig. 1.
Example of an SP population from an unenriched whole bone marrow sample. In order tovisualize the characteristic SP pattern of bone marrow cells, the emission of Hoechst 33342must be displayed bimodally as Hoechst Red vs. Hoechst Blue, both in a linear scale. Thecells concentrated at the lower left corner represent red blood cells and cellular debris, whilethe rest of the sample is mainly grouped on the upper right side of the acquisition window.The SP gate is drawn around the tail that diagonally emerges from the main population andusually represents 0.02–0.05% of whole bone marrow cells.
Methods Mol Biol. Author manuscript; available in PMC 2013 April 10.
Rossi et al.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptPage 10
Fig. 2.
Sorting strategy for SP KLS (SP c-Kit + Lineage− Sca-1+) cells. (a) SP gate: the first stepconsists in displaying the Hoechst 33342 efflux pattern is linear mode (as Hoechst Red vs.Hoechst Blue) and gating the SP population. (b) Morphological characteristics: display theSP cells gated in the first panel as FSC (forward scatter) vs. SSC (side scatter) and draw asecond gate as shown in figure. (c) Lineage staining (PE-Cy5): gate out cells that expressmarkers of hematopoietic terminal differentiation and select Lineage-negative cells. (d) c-Kit vs. Sca-1: the last panel shows the expression of the stem cell markers c-Kit (PE) andSca-1 (FITC) in SP/Lineage-negative cells. This is the sorting gate, comprising the SP KLSpopulation. (e–h) The panels on the right show, by comparison, how unenriched bone
marrow cells (gated only on the live population from (e)) distribute on the same parameters.
Methods Mol Biol. Author manuscript; available in PMC 2013 April 10.
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