Journal of Immunological Methods 310 (2006) 171–181www.elsevier.com/locate/jim

Research paper

Out of the blue: A comparison of Hoechst side population (SP)analysis of murine bone marrow using 325, 363 and 407 nm

excitation sources☆

Catherine Simpson a, Daniel J. Pearce b,1, Dominique Bonnet b, Derek Davies a,⁎

a FACS Laboratory, London Research Institute, Cancer Research UK, 44 Lincoln's Inn Fields, London, WC2A 3PX, United Kingdomb Hematopoietic Stem Cell Laboratory, London Research Institute, Cancer Research UK, London, WC2A 3PX, United Kingdom

Received 26 October 2005; received in revised form 22 December 2005; accepted 11 January 2006Available online 7 February 2006

Abstract

Background: The discrimination of side population (SP) cells is a useful flow cytometric tool for the study of putative stem cells inmany tissue types. Previous studies on murine bone marrow (BM) SP cells and concurrent surface immunophenotyping provide agood model for comparing Hoechst bivariate profiles. Here we compare the results of SP cells from the same specimens generatedon three different instruments.Methods: Concurrent SP analysis and immunophenotyping was performed on murine BM. The data acquisition was performed ona DakoCytomation MoFlo, a Becton Dickinson LSR and a Becton Dickinson FACSAria to compare the ability of each instrumentto discriminate putative stem cells. Further experiments examined the effect of apoptotic changes, assessed by Annexin V binding,and the presence of nucleated erythroid precursors on the Hoechst profile.Results: Identifiable and equivalent side populations were generated on all three instruments despite differences in appearance ofthe Hoechst profiles. Differences in laser type, collection filter combinations and the presence of erythroid precursors and apoptoticcells all contributed to this effect.Conclusions: In our hands, murine SP cells with the expected surface immunophenotype can be identified on a MoFlo, LSR and afactory standard FACSAria.© 2006 Elsevier B.V. All rights reserved.

Keywords: Hoechst; Side population; KTLS; Instrumentation; Apoptosis

☆ A preliminary report of these data was presented at the XXII ISACmeeting (Montpellier, May 2004). This work was supported by CancerResearch UK and an Association for International Cancer Researchgrant No. GA3160 to D. Bonnet.⁎ Corresponding author. Tel.: +44 20 72693394; fax: +44 20

72693479.E-mail address: derek.davies@cancer.org.uk (D. Davies).

1 CS and DJP contributed equally to this work.

0022-1759/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.jim.2006.01.005

1. Introduction

Much of the work on the investigation of stem cellsinvolves functional assays where identification andisolation of these rare cells in a functionally uncompro-mised state is essential. The characterisation ofhematopoietic stem cells (HSCs) is well establishedusing multiparameter analysis of single cell suspensionsby flow cytometry. Combinations of surface markershave been successfully used to isolate stem cells in the

172 C. Simpson et al. / Journal of Immunological Methods 310 (2006) 171–181

absence of unique epitopes (Wolf et al., 1993). Thisoften involves the identification of immature cellsthrough a lack of expression of epitopes associatedwith differentiation. Physiological differences have alsobeen exploited and avoid the potential receptoractivation associated with immunophenotyping. Onedifference that has been well documented is the abilityof HSCs to efflux the DNA binding dye Hoechst 33342(Wolf et al., 1993). Increased numbers of ATP-binding-cassette (ABC) transporters have been shown to beresponsible for this phenomenon. Comparison ofmessenger RNA (mRNA) levels in bone marrow forthe three major efflux pumps MDR1 (P-glycoprotein,ABCB1), multidrug resistance associated protein 1(MRP1, ABCC1) and breast cancer resistant protein(BCRP ABCG2), reveals that ABCG2 is the predom-inant form in these cells (Zhou et al., 2001; Scharenberget al., 2002; Sarkadi et al., 2004). Goodell et al.described a so-called side population (SP) after stainingwhole murine bone marrow (BM) with Hoechst 33342(Goodell et al., 1996). The SP cells were able to effluxthe dye using ABC transporters and formed a distinctpopulation of cells with reduced Hoechst staining whenanalyzed by flow cytometry on a bivariate dot plotshowing Hoechst red (abscissa) versus Hoechst blue(ordinate) emissions. These cells were subsequentlyshown to have a high repopulation potential, withsuccessful engraftment achieved in some cases with justone cell (Matsuzaki et al., 2004). Since then, SP cellshave been described in many tissues other than bonemarrow, e.g., breast, prostate, skin and liver (Alvi et al.,2003; Bhatt et al., 2003; Bhattacharya et al., 2003;Terunuma et al., 2003; Wulf et al., 2003) and SP stainingprotocols vary slightly to account for different cell types.

The quality of stoichiometric DNA staining byHoechst has been shown to be dependent on cell type,cell concentration, dye concentration, pH, temperatureand time (Szabo et al., 1981; Smith et al., 1985; Steenand Stokke, 1986). Increased red fluorescence is seenwith increased concentrations of Hoechst and a similarred shift has also been reported in apoptotic and deadcells (Ellwart and Dormer, 1990; Petersen et al., 2004).The latter is poorly understood but is thought to be theresult of changes in DNA conformation that in turnaffect dye–dye interactions and fluorescence emissioncharacteristics. In addition to the shift in fluorescence,apoptotic cells undergo subtle membrane changes thatallow an increased rate of Hoechst uptake (Szabo et al.,1981; Ormerod et al., 1992). Both phenomena can beexploited in the presence of propidium iodide (PI) toidentify apoptotic cells. Dead cells lose their membraneintegrity and as a result are positive for PI. The PI

negative cells that remain in the region of increased redfluorescence and/or take up the dye at a faster rate havebeen shown to be apoptotic (Ellwart and Dormer, 1990;Ormerod et al., 1992).

Sourcing the appropriate instrumentation for SPidentification is also important. As Hoechst is maxi-mally excited by UV wavelengths (excitation maximum350 nm), assessment of stained cells was initiallyrestricted to stream-in-air sorters equipped with power-ful water-cooled argon ion lasers. The collection opticsof these instruments are less efficient than systems thatuse flow cells and gel-coupled optics. Therefore highUV laser powers (around 60 mW) are required toadequately excite the Hoechst and allow the detection ofthe subtle changes in emission intensity that areobserved in SP identification.

Recent developments in laser technology have seenthe introduction of numerous solid-state low powerlasers on the market with UV or near-UV wavelengths(Shapiro and Perlmutter, 2001). While the lower laserpower levels restrict their use for stream-in-air sorterstheir potential for use in systems with more efficientemission collection optics is obvious. Hoechst SPidentification using violet and near-UV laser diodes ona modified bench top instrument has recently beenpublished (Telford and Frolova, 2004). Assessment ofthe near-UV sources showed excellent Hoechst SPresolution while the 401 and 408 nm violet lasers weredeemed poor at resolving SP cells (Telford and Frolova,2004).

We have used three instruments in our laboratory thathave the potential to identify Hoechst SP cells. These are:a MoFlo cell sorter (DakoCytomation, Fort Collins, CO)equipped with an argon ion laser (Coherent Innova 70 C-5) tuned to mid-range UV (333.6 to 363.8 nm); an LSR(Becton Dickinson, San Jose, CA) with the Europeanbeam-key switch modification and helium–cadmium(Kimmon Electric IK series) UV laser (325 nm); and aFACSAria (Becton Dickinson, San Jose, CA) equippedwith a Point Source 407 nm violet laser (Table 1). Theidentification of murine BM SP populations has beenwell established in our laboratory using the MoFlo andassessment of the relationship between SP populationsand surface immunophenotyping has recently beenpublished (Pearce et al., 2004). Cells that lack maturedifferentiation markers (lineage negative) and express c-kit, Sca-1 and are Thy-1 dim (KTLS) have been shown tohave a high repopulation potential (Osawa et al., 1996).It has also been demonstrated that this phenotype incombination with SP results in a 2800 fold enrichmentfor repopulating units in long-term competitive in vivoassays (Pearce et al., 2004). This model therefore allows

Table 1Summary of flow cytometer configurations

MoFlo FACSAria LSR

Laser type Argon gas Violetsolid-state

HeliumCadmium gas

Laser manufacturer/model

Coherentinnova70C-5

Pointsource

Kimmonelectric IKseries

Laser wavelength(nm)

333.6–363.8 407 325

Laser power (mW) 60 10 8Hoechst blue

filter (nm)424/44 450/40 424/44

Hoechst red filter (nm) 620 LP 620 LP 620 LPDichroic (nm) 610 DCLP 595 DCLP 630 DCLPEfficiency of Hoechst

excitation at laserwavelength (%)

100 3 54

Efficiency of PIexcitation at laserwavelength (%)

100 9 100

Displayed in tabular format are the configuration data for the threeflow cytometers compared in this study. Laser power is given inmilliWatt (mW). LP=long-pass. DCLP=dichroic long pass. Fluores-cence efficiencies are from www.probes.com/servlets/spectra.

173C. Simpson et al. / Journal of Immunological Methods 310 (2006) 171–181

a phenotypic comparison of murine BM SP profilesgenerated on different instruments.

Here, we compare the Hoechst bivariate plotsgenerated by all three instruments and assess the affectsof apoptotic cells and erythroid precursors on theappearance of the profiles. Concurrent surface immu-nophenotyping is used to assess the ability of aFACSAria and LSR to detect murine BM SP cellsusing the MoFlo as a laboratory benchmark.

2. Materials and methods

2.1. Mice

All experiments were performed in compliance withHome Office and institutional guidelines. C57Bl6-Thy-1.1(Ly5.2) mice colonies were maintained in our animalfacility, but were originally obtained from JacksonLaboratories. C57Bl6 mice aged 8–12 weeks were usedfor all experiments in this study.

3. Cell preparation

Bone marrow was flushed from C57B16 murinefemurs and tibias and red blood cells were lysed withammonium chloride (Stem Cell Technologies, Vancou-ver, BC, Canada). Nucleated cells were labelled withHoechst 33342 (Sigma Chemical Co. St. Louis MO) aspreviously described (Sarkadi et al., 2004). Briefly, cells

were resuspended at 106 cells per ml in pre-warmedDMEM containing 2% FCS, 10 mM HEPES, and 5 μgper ml Hoechst 33342 and incubated at 37 °C for 90min. Reserpine (Sigma Chemical Co. St. Louis MO)was used at a final concentration of 5 μM as aninhibitory control (Zhou et al., 2001). Once stained, thecells were maintained at 4 °C during antibody labellingand until analysis.

Antibody labelling was performed in phosphate-buffered saline (PBS), 2% fetal calf serum (FCS), 10mMHEPES for 30 min against appropriate matched isotypecontrols. The following antibodies were used (all fromPharmingen, SanDiego, CA): fluorescein isothiocyanate(FITC) conjugated anti-Thy-1, CD45; phycoerythrin(PE) conjugated anti-CD5, CD11b, B220, Ter-119, andGr-1; biotinylated anti-Sca-1 (subsequently labelled withPE-Cy5 conjugated streptavidin); and allophycocyanin(APC) conjugated anti-CD117 (c-Kit). The lineagecocktail used contained PEconjugated anti-CD5,CD11b, B220, Gr-1 and Ter-119 antibodies. Stainedcells were resuspended in PBS, 2% FCS, 10 mMHEPES, 2 μg/ml PI and passed through a 70 μm meshbefore analysis.

3.1. Annexin V labelling

For time course experiments, 100 μl of 10× AnnexinV Binding Buffer (BD Pharmingen, San Jose, CA) wasadded to 900 μl of the resuspended cells and mixed.Then 5 μl of directly conjugated Annexin V AlexaFluor® 647 (Molecular Probes, Leiden, The Nether-lands) were added to the cells before incubation at 37 °Cfor 15 min. This was to allow Annexin V binding beforethe addition of 5 μg/ml Hoechst at time zero. Aliquots of200 μl were removed at 15 min intervals for 90 min and2 μg/ml PI was added to the tubes as above prior toanalysis and collected through a 580/30 BP filter withlogarithmic amplification after excitation by the 488 nmlaser. Alexa Fluor® 647 was excited by the HeliumNeon (HeNe) laser and fluorescence collected withlogarithmic amplification through a 660/20 BP filter. Allincubations were performed at 37 °C in the dark anddata acquisition was performed on a MoFlo cell sorter.

4. Flow cytometry

4.1. MoFlo

This instrument has the Z-configuration and isequipped with three lasers. There are two CoherentINNOVA argon lasers, the primary tuned to 488 nm andthe secondary to mid range UV (333.6 to 363.8 nm) and

174 C. Simpson et al. / Journal of Immunological Methods 310 (2006) 171–181

a Helium Neon laser (Spectra Physics) emitting at633 nm. The 488 nm laser was adjusted to a poweremission of 200 mW, the UV laser 60 mWand the HeNeemits 35 mW. Emission from UV interrogation wascollected through the second pinhole and split through a610 nm long pass dichroic mirror (DCLP) to a 620 nmlong pass (LP) filter for the Hoechst red collection and a424/44 nm band pass (BP) filter for the Hoechst bluecollection (Fig. 1A).

4.2. FACSAria

This instrument is a factory standard equipped withthe optional 10 mW Violet (407 nm) solid-state laser.Some optical filter changes were made so that the violetline emission is split through a 595 nm DCLP with theHoechst red collected through a 620 nm LP filter and theHoechst blue through a 450/40 nm BP filter (Fig. 1B).

4.3. LSR

This instrument has been modified with a Europeanbeam-key switch. In this modification, the light path hasbeen altered so that signals generated by the UV laserare steered by a mirror rather than by a longpass dichroicfilter to the relevant detectors. In this way wavelengthsabove 520 nm may be measured. The UV line isgenerated from an 8 mW helium–cadmium gas laserwith a wavelength of 325 nm. UV emission is split

Hoechst Red

Hoech

620 LP

595 DCLP M

450/40

Hoechst Red

Hoechst Blue

620 LP

424/44 BP

610 DCLP

MoFloA B

Fig. 1. Diagrammatic representation of flow cytometer configuration. MoFlo610 nm DCLP to a 620 nm LP filter for the Hoechst red collection and a 424/Hoechst emission is split via a 595 nm DCLP. The Hoechst red collected throfilter. LSR configuration (C): UVemission is split through a 630 nm DCLP wblue is passed through a 424/44 nm BP filter. In all cases the black arrow re

through a 630 nm DCLP with the Hoechst red collectedthrough 620 nm LP filter in the P6 (FL5) position andthe Hoechst blue is passed through a 424/44 nm BP filterto the P5 (FL4) position (Fig. 1C). Application of thismodification means that it is not possible to use the UVand red lasers simultaneously and it is important to closethe shutter on the HeNe laser when analysing cells forSP. Full KTLS assessment could therefore not be testedon the LSR with the fluorophores of choice for thisreason. However, KLS and SP profiles from the LSRhave previously shown similar results when comparedto the MoFlo (data not shown).

For SP analysis, at least 1×105 total events werecollected and all subsequent analysis was performedusing FlowJo software (Tree Star Inc. Ashland OR).

5. Results

Hoechst 33342 is a cell-permeable DNA-specificbisbenzimidazole dye with a preference for sequences ofthree A–T base pairs. It is excited by UV wavelengths(max 350 nm) and has an emission maximum of 470 nm(Shapiro, 2003). For SP analysis, blue fluorescenceemission (424/44 nm BP) and red fluorescence emission(620 nm LP) are measured simultaneously. The SPprofile can be seen on a bivariate dot plot of Hoechstemission, with linear red collection on the abscissa andlinear blue on the ordinate (example in Fig. 2A). Debrisand residual mature red blood cells are Hoechst negative

st Blue

irror

BP

Hoechst Red

Hoechst Blue

620 LP

424/44 BP

630 DCLP

FACS Aria LSRC

configuration (A): Emission from UV interrogation was split through a44 nm BP filter for the Hoechst blue collection. Aria configuration (B):ugh a 620 nm LP filter and the Hoechst blue through a 450/40 nm BPith the Hoechst red collected through 620 nm LP filter and the Hoechstpresents direction of the total Hoechst emission.

0 50000 100000 150000 200000 2500000

50000

100000

150000

200000

250000

0.075

0 1000 2000 3000 40000

1000

2000

3000

4000

0.062

0 200 400 600 800 10000

200

400

600

800

1000

0.056

Hoechst Red

Forward scatterH

oech

st B

lue

Hoe

chst

Blu

e

Hoe

chst

Blu

eSi

de s

catte

r

Side

sca

tter

Side

sca

tter

MoFlo FACS Aria LSR

BA

0 1000 2000 3000 40000

1000

2000

3000

4000E

0 200 400 600 800 10000

200

400

600

800

1000

0 1000 2000 3000 40000

1000

2000

3000

4000

FD

C

Fig. 2. Comparison of SP profiles between flow cytometers. (A, B and C) Representative Hoechst red vs. Hoechst blue SP profiles of murine bonemarrow, after gating on live (propidium iodide negative) events, from MoFlo, Aria and LSR, respectively. Although there are subtle differencesbetween the plots, the SP is clearly visible in all cases. (D, E and F) Scatter characteristics of SP cells (black dots) analysed on the MoFlo, Aria andLSR, respectively. SP cells identified on all three machines possess scatter characteristics that are typical of stem/progenitor cells in murine bonemarrow.

175C. Simpson et al. / Journal of Immunological Methods 310 (2006) 171–181

and appear towards the origin. Propidium iodide is alsoexcited by UV wavelengths and dead cells, which arepositive for the dye, fluoresce strongly red. This allowsdead and unstained cells to be excluded from theanalysis.

We had already observed a degree of flexibility infilter selection for SP analysis and this has also beendocumented in the literature (Telford and Frolova,2004). We used 424/44 nm BP filters for blue collectionon the LSR and MoFlo and 450/40 nm BP on theFACSAria. The 450/40 nm BP was selected for theFACSAria as the 424/44 nm BP would allow transmis-sion of the violet excitation wavelength of 407 nm.The long pass filters used to collect the Hoechstred fluorescence were the same on all instruments(620 nm LP).

On three separate occasions, the same Hoechststained murine BM preparation was acquired on allthree instruments on the same day. Representativeprofiles are shown in Fig. 2A–C. Although the totalHoechst emission profiles vary between the three

instruments, the side populations are clearly identifiablein all cases with comparable percentages. When SPevents from all three instruments were back-gated ontoforward and side scatter dot plots, the low scattercharacteristics of murine stem/progenitor cells wereobserved (Fig. 2D–F). In each case, samples treatedwith reserpine showed absence of the SP. Cell viabilitywas similar between instruments and consistent with ourprevious observations (Pearce et al., 2004).

We then wanted to confirm that the surfaceimmunophenotype was that expected for KTLS cells.When SP cells are back-gated onto bivariate dot plotsshowing combinations of these surface markers, theyexpress the same staining patterns (Fig. 3). The datafrom the MoFlo and FACSAria were more specificallyanalyzed for KTLS (Fig. 4). The lineage negative, Sca-1positive cells were gated onto c-Kit, Thy-1 plots andresults from both instruments were comparable (Fig. 4Cand F). Back-gating the SP events onto these templatesshowed that most of the cells fell within the KTLS gate.This confirmed that the SP cells discriminated by both

100

100

101

101

102

102

103

103

104

104100

100

101

101

102

102

103

103

104

104

100

100

101

101

102

102

103

103

104

104100

100

101

101

102

102

103

103

104

104

100

100

101

101

102

102

103

103

104

104

105

105

101

101

102

102

103

103

104

104

105

105

101

101

102

102

103

103

104

104

105

105

101

101

102

102

103

103

104

104

Lineage-PE

Lineage-PE

Lineage-PE

MoFlo

LSR

FACSAria

Thy

-1-F

ITC

Sca-

1-PE

Cy5

c-K

it-A

PC

A C

D F

G

B

E

H

Fig. 3. Immunophenotypic comparison of SP profiles between flow cytometers. Representative analysis of the immunophenotype of cells identifiedas SP on MoFlo (A–C), FACSAria (D–F) and LSR flow cytometers (G and H). SP cells (dots) are overlaid onto density plots of the total livenucleated cell bone marrow population. All instruments show similar expression of Thy-1 (A,D,G) and Sca-1 (B,E,H); the MoFlo and FACSAriashow similar expression of c-Kit (C and F). Because c-Kit was conjugated to APC, this combination could not be used on the LSR in conjunction withtwo parameters from the UV line.

176 C. Simpson et al. / Journal of Immunological Methods 310 (2006) 171–181

instruments had a surface immunophenotype consistentwith long-term repopulating cells. Consistent with ourprevious study, we did not observe a significantassociation between the percentage of KTLS cells andthe extent of Hoechst efflux (Pearce et al., 2004).

The most notable difference between instrumentswhen assessing the Hoechst bivariate profiles is not theSP, but a population of cells with reduced Hoechst blueand increased Hoechst red fluorescence (referred to hereas Hoechst Redhi). This Hoechst Redhi population ismore obvious on the MoFlo profiles than those from theLSR and is completely absent on the FACSAria plots(see arrows on Fig. 2A and C). We had previously

observed significant variations in this population duringassessment of different murine BM preparations withour standard instrument set up on the MoFlo. Variationsbetween instruments can in part be attributed todifferences in excitation wavelength and collection filtercombinations. Although the resolution of the SP profileappears unaffected by these variations, we wanted toinvestigate the Hoechst Redhi population further.

6. Assessment of apoptosis

Apoptotic cells undergo changes in DNA conforma-tion that cause an increase in red fluorescence when

100 101 102 103 104100

101

102

103

104

100 101 102 103 104100

101

102

103

104

100 101 102 103 104100

101

102

103

104

105101 102 103 104

105

101

102

103

104

105101 102 103 104

105

101

102

103

104

105101 102 103 104

105

101

102

103

104

0.6A

54.2

B

91.8

CLin- Sca-1+ SP

MoFlo

0.6

D

81

F

51.4

E Lin- Sca-1+ SP

FACSAria

Lineage-PE

Lineage-PE c-Kit-APC

Sca-

1-PE

Cy5

Sca-

1-PE

Cy5

Thy

-1-F

ITC

Thy

-1-F

ITC

Thy

-1-F

ITC

Thy

-1-F

ITC

c-Kit-APC

c-Kit-APC c-Kit-APC

Fig. 4. Comparison of stem cell immunophenotype of SP profiles between flow cytometers. Representative analysis of SP cells from MoFlo (A–C)and FACSAria (D–F) flow cytometers. The live population was first gated for lineage negative and Sca-1+ cells (A and D). These cells were thengated for c-Kit+ and Thy-1+ for the final KTLS population (B and E). The SP cells from both instruments were back gated onto the same dot plots andfall in the same region confirming that the SP cells have the KTLS phenotype (C and F).

177C. Simpson et al. / Journal of Immunological Methods 310 (2006) 171–181

stained with Hoechst (Ellwart and Dormer, 1990). Thesecells also show membrane changes that allow anincreased rate of Hoechst uptake whilst still excludingPI (Ormerod et al., 1992). Time-course studies showed apopulation of PI negative cells staining with Hoechst asearly as 15 min. This eventually resolved as the HoechstRedhi population while the rest of the cells required 90min for stoichiometric staining (Fig. 5). In addition tovariations in Hoechst uptake, apoptotic cells undergo amembrane change whereby phosphatidylserine residuesthat normally reside at the inner cytoplasmic membraneare translocated to the external membrane. Theseresidues can then be detected by the specific bindingprotein Annexin V (Koopman et al., 1994). The HoechstRedhi population was assessed for this relocation bystaining the cells with Annexin V directly conjugated toAlexa Fluor® 647. The analysis revealed that theAnnexin V positive, PI negative apoptotic cells werelocated in the Hoechst Redhi region (Fig. 5C and F), theproportion varying between preparations from less than5% to greater than 40% (data not shown). Time course

studies showed that the early staining Hoechst popula-tion that appeared after 15 min was also Annexin Vpositive (Fig. 5C and F).

7. Nucleated red blood cell staining

Blue light emitted by UV-excited Hoechst stainedcells has been shown to be reabsorbed by hemoglobinand cause a reduction in the intensity of blue fluores-cence (Stokke et al., 1990). We therefore speculated thatnucleated erythroid precursors may also contribute to theHoechst Redhi population. Staining BM with theerythroid marker Ter119 and the pan-leukocyte markerCD45, confirmed that is indeed the case with all cells thatare positive for Ter119 and negative for CD45 located inthis region (Fig. 6D and F). In addition, a group of cellsshowing intermediate CD45 expression were also found(Fig. 6B). When back gated, this population was shownto have discrete forward and side scatter characteristicsand, while interesting, further immunophenotyping wasnot pursued. Other investigators have noticed

14.1

Hoechst Red Hoechst Red

Hoe

chst

Blu

e

Hoe

chst

Blu

e

Annexin V Alexa 647

Time 90 minutes

0 1000 2000 3000 40000

1000

2000

3000

4000

0 1000 2000 3000 40000

1000

2000

3000

4000

100 101 102 103 104100

101

102

103

104

100 101 102 103 104100

101

102

103

104

2020

Hoechst Red Hoechst RedH

oech

st B

lue

Hoe

chst

Blu

e

Annexin V Alexa 647

Prop

idiu

m I

odid

eTime 15 minutes

A

D

Prop

idiu

m I

odid

e

0 1000 2000 3000 40000

1000

2000

3000

4000

0 1000 2000 3000 40000

1000

2000

3000

4000

C

F

B

E

Fig. 5. Analysis of Hoechst Redhi cell population and apoptotic cells. Immunophenotypic analysis of Hoechst profiles at 15 min (A–C) and 90 min(D–F). Identification of the PI-negative Annexin-V+ apoptotic population (A and D). After 15 min incubation, the Hoechst Redhi population appears(B) and persists after stoichiometric staining at 90 min (E). Apoptotic PI negative Annexin-V+ cells (black dots) are found within the Hoechst Redhi

populations (C and F).

178 C. Simpson et al. / Journal of Immunological Methods 310 (2006) 171–181

pronounced differences in the Hoechst staining char-acteristics of murine lymphocytes and this supports thepossibility of small populations of viable nucleatedCD45 positive cells also contributing to the HoechstRedhi region (Shapiro, 2003).

8. Discussion

The identification and isolation of rare stem cells in afunctionally uncompromised state is important forcontinued research. In most cases there is no specificmarker to target stem cells so combinations of surfacemarkers are used for identification. Exploiting physio-logical differences in these cells is an attractivealternative as antibodies to surface epitopes may triggersignalling with unknown downstream effects. Stem cellshave large numbers of membrane transporters andHoechst DNA dye has been shown to be an appropriatesubstrate for these (Wolf et al., 1993; Zhou et al., 2001).Strict staining protocols that control cell and dyeconcentrations, pH, temperature and time, can reveal

a population of cells that have low Hoechst uptake byvirtue of their ability to remove much of the dyethrough active transport (Goodell et al., 1996). TheseSP cells are optimally identified on a bivariate plotwith linear Hoechst red on the abscissa and linearHoechst blue on the ordinate and have been welldefined in murine BM (Pearce et al., 2004). SP cellshave also been identified in other tissues and modifica-tions of the staining conditions have been made toaccount for differences in cell type and their effect onstaining dynamics (Alvi et al., 2003; Bhatt et al., 2003;Bhattacharya et al., 2003; Terunuma et al., 2003; Wulfet al., 2003).

We have observed heterogeneity between differentpreparations of murine BM when assessing Hoechstbivariate profiles and these were highlighted furtherwhen we acquired data on three different instruments.Our main aim was to assess the ability of the FACSAriato discriminate a side population using a 407 nm solid-state laser. The identification of murine BM SP cells andsurface KTLS immunophenotyping has been well

0 1000 2000 3000 4000 0 1000 2000 3000 4000 0 1000 2000 3000 4000

CD 45-PE-Cy7

Hoechst Red

Hoechst Red

Hoe

chst

Blu

e

Hoe

chst

Blu

eTer 119-PE

Side

sca

tter

0

1000

2000

3000

4000

0

1000

2000

3000

4000

7.797.79

100 101 102 103 104 100 101 102 103 104

Ter 119 positive

A

Side

sca

tter

0

1000

2000

3000

4000

0

1000

2000

3000

4000

Hoe

chst

Blu

e

0

1000

2000

3000

4000

0 1000 2000 3000 4000 0 1000 2000 3000 4000 0 1000 2000 3000 4000

Hoe

chst

Blu

e

Hoe

chst

Blu

e

0

1000

2000

3000

4000

0

1000

2000

3000

4000

Hoe

chst

Blu

e

0

1000

2000

3000

4000

CD45 negative

CD45intermediate

CD 45 high

B

C

11.2

6.36

79.5

Ter 119 positiveD

CD 45 negativeF CD 45 intermediateG CD 45 highH

CD 45 positiveE

Fig. 6. Analysis of Hoechst Redhi cell population in relation to Ter-119 and CD45. Identification of the Ter-119+ erythroid cell population for furtheranalysis (A). A CD45 vs. side scatter plot with CD45-negative, intermediate and high populations indicated (B). Hoechst red vs. blue SP plot of thetotal live population (C). Ter-119+ cells (black dots) overlaid onto the total live population (D). Ter-119+ cells are mostly restricted to the HoechstRedhi region. Total CD45+ (black dots) overlaid onto the total live cell population (E). Cells expressing negative (F), intermediate (G) and high (H)levels of CD45 (black dots) overlaid onto the total live population. Part of the CD45 intermediate population falls within the Hoechst Redhi region.

179C. Simpson et al. / Journal of Immunological Methods 310 (2006) 171–181

established in our laboratory using the MoFlo (Pearce etal., 2004). This model therefore allows phenotypiccomparisons of murine BM SP profiles generated fromother instruments.

The total Hoechst emission profiles do vary betweenthe three instruments but the side populations are clearlyidentifiable in all cases (Fig. 2A–C). Differences in laserexcitation wavelengths, laser power, excitation efficien-cies of the Hoechst and collection optics all contribute to

the variations seen. When analyzing Hoechst on theFACSAria, the 407 nm line has an excitation efficiencyof approximately 3% (Table 1). Acquisition of a DNAprofile is possible only because of the efficacy of theflow cell and collection optics.

Despite the differences in the shape of the profiles,backgating the SP populations onto scatter dot plotsgenerated comparable, low scatter profiles (Fig. 2D–F).Concurrent surface immunophenotyping of the SP cells

180 C. Simpson et al. / Journal of Immunological Methods 310 (2006) 171–181

showed similar staining patterns on all instruments (Fig.3). Specific KTLS analysis of the FACSAria SPpopulations showed a phenotype matching the SPcells from the MoFlo (Fig. 4). This confirms that thecells from the SP gates have a surface immunopheno-type consistent with long term repopulating cells(Osawa et al., 1996; Pearce et al., 2004).

One of the most notable differences between theprofiles generated by each instrument was the presenceof a Hoechst Redhi population showing reduced Hoechstblue and increased Hoechst red fluorescence. Thispopulation was most obvious on the MoFlo andvirtually absent on the FACSAria profiles and thevariations can be attributed mostly to differences inexcitation wavelength and collection filter combina-tions. A previous study (Eaker et al., 2004) had foundthat there were noticeable differences between SPprofiles obtained using ultraviolet and violet excitationsources and we had previously observed differencesbetween murine BM experiments on the MoFlo. Whilethe resolution of the SP profiles appears unaffected bythese variations, we wished to explore this phenomenonfurther.

The uptake of Hoechst and its emission wavelengthsare affected by cell type, cell concentration, dyeconcentration, temperature, pH and time (Alvi et al.,2003; Bhatt et al., 2003; Matsuzaki et al., 2004) and therecent paper by Petersen et al. has specifically addressedthe spectral changes associated with different intracel-lular dye concentrations (Petersen et al., 2004). Changesin DNA conformation such as those associated withapoptosis can also affect the spectral characteristics ofHoechst and results in a shift in red fluorescence(Ellwart and Dormer, 1990). Probing the Hoechst Redhi

population for membrane changes associated withapoptosis showed Annexin V positive staining. Theexternal presentation of phosphatidylserine whilstmaintaining membrane integrity is one of the hallmarksof early apoptosis (Shapiro, 2003). Time courseanalyses showed these cells stained with Hoechst after15 min while the Annexin V negative population took90 min to reach stoichiometric staining (Fig. 5). Theapoptotic cells present in these preparations were notspecifically induced and proportions varied betweenexperiments from less than 5% to greater than 40%. Thisvariation is probably the result of a combination ofpossible effects including the stress of extraction of theBM from the femurs and tibias, different reagent lotsand variations in time between extraction, staining andacquisition.

This spectrum of apoptotic cells showing membraneand DNA conformational changes only accounted for

part of the Hoechst Redhi region and again thisproportion varied between preparations. It has beenpreviously shown that hemoglobin has the ability toreabsorb blue fluorescence emission of Hoechst, result-ing in a quenching of the fluorescence (Stokke et al.,1990). Small numbers of red blood cells will have nogreat effect as these anucleate cells are dye negative andappear in the origin of the Hoechst bivariate profile.However, nucleated erythroid precursors present inwhole BM preparations may have some interestingconsequences. The close proximity of hemoglobin to aHoechst positive nucleus may result in a reduction ofblue fluorescence. We therefore speculated that thepresence of nucleated erythroid precursors in BMpreparations might also contribute to the HoechstRedhi population.

Staining murine BM with the red cell marker Ter119did locate nucleated precursors in this region (Fig. 6D).Concurrent staining with Ter119 and CD45 showedTer119 positive and PI negative cells formed part of theHoechst Redhi population. In addition, cells withintermediate expression of CD45 were also located inthis region and were shown to have discrete forward andside scatters. In the absence of Annexin V positive cells,the resultant shift in the spectral emission of these cellsis most likely due to inherent differences in DNAconformation that are not associated with apoptosis.Other investigators have noted pronounced differencesin the staining of murine lymphocytes and haveexploited this phenomenon to identify subsets (Shapiro,2003).

The proportions of different cell types and theamount of apoptosis will vary between different murineBM preparations and may account for the previouslyobserved variations in the Hoechst Redhi population.While these cells cannot be identified on FACSAriabivariate plots primarily due to the differences in filtercombinations and laser excitation wavelength, they haveno bearing on the identification of SP cells. Theseobservations do, however, strongly support an elementof caution in interpretation of Hoechst bivariate profileswhen assessing data generated from heterogeneoussamples. In a heterogeneous population, cells withdifferent morphology and DNA conformations mayhave different staining dynamics and emission char-acteristics and could have the potential to affectinterpretation of data.

While it could be argued that the murine BM SPprofile from the FACSAria is not as well resolved as onthe MoFlo, we have shown that each cytometeridentifies the same cells with a profile discrete enoughto enable surface immunophenotyping and, importantly,

181C. Simpson et al. / Journal of Immunological Methods 310 (2006) 171–181

sorting. Although we have previously shown that SPcells sorted on the basis of KTLS positivity have thehighest repopulation potential (Pearce et al., 2004),future studies will involve confirmation that thepopulation identified on the FACS Aria and other lightsources has the same potential.

In all cases of SP identification, it is important thatthe cytometer is clean, well-aligned and checked forsensitivity; in the case of a violet diode, it is alsoimportant to bear in mind that the quoted emittedwavelength given by the manufacturer will have atolerance of ±5 nm and the lower the true wavelengththe better the excitation of the Hoechst dye. Resolutionof the SP is dependent on the laser wavelength and thelaser power — if possible the power output of the laserclose to the interrogation point should be checked.Identification of an SP population on the FACSAria hasthe potential to increase the flexibility with regard tohardware necessary to identify these cells, at least inmurine bone marrow.

Acknowledgements

The authors would like to acknowledge the technicalassistance of Chris Ridler (Haematopoietic Stem CellLaboratory, Cancer Research UK), and to thank KirstyAllen, Ayad Eddaoudi and Gary Warnes, (FACSLaboratory, Cancer Research UK) for their cytometricexpertise and helpful discussions.

References

Alvi, A.J., Clayton, H., Joshi, C., Enver, T., Ashworth, A., Vivanco,M.M., Dale, T.C., Smalley, M.J., 2003. Functional and molecularcharacterisation of mammary side population cells. Breast CancerRes. 5, R1.

Bhatt, R.I., Brown, M.D., Hart, C.A., Gilmore, P., Ramani, V.A.,George, N.J., Clarke, N.W., 2003. Novel method for the isolationand characterisation of the putative prostatic stem cell. Cytometry54A, 89.

Bhattacharya, S., Jackson, J.D., Das, A.V., Thoreson, W.B., Kus-zynski, C., James, J., Joshi, S., Ahmad, I., 2003. Directidentification and enrichment of retinal stem cells/progenitors byHoechst dye efflux assay. Invest. Ophthalmol. Vis. Sci. 44, 2764.

Eaker, S.S., Hawley, T.S., Ramezani, A., Hawley, R.G., 2004.Detection and enrichment of hematopoietic stem cells by sidepopulation phenotype. Methods Mol. Biol. 263, 161.

Ellwart, J.W., Dormer, P., 1990. Vitality measurement using spectrumshift in Hoechst 33342 stained cells. Cytometry 11, 239.

Goodell, M.A., Brose, K., Paradis, G., Conner, A.S., Mulligan, R.C.,1996. Isolation and functional properties of murine hematopoieticstem cells that are replicating in vivo. J. Exp. Med. 183, 1797.

Koopman, G., Reutelingsperger, C.P., Kuijten, G.A., Keehnen, R.M.,Pals, S.T., van Oers, M.H., 1994. Annexin V for flow cytometric

detection of phosphatidylserine expression on B cells undergoingapoptosis. Blood 84, 1415.

Matsuzaki, Y., Kinjo, K., Mulligan, R.C., Okano, H., 2004.Unexpectedly efficient homing capacity of purified murinehematopoietic stem cells. Immunity 20, 87.

Ormerod, M.G., Collins, M.K., Rodriguez-Tarduchy, G., Robertson,D., 1992. Apoptosis in interleukin-3-dependent haemopoieticcells. Quantification by two flow cytometric methods. J. Immunol.Methods 153, 57.

Osawa, M., Nakamura, K., Nishi, N., Takahasi, N., Tokuomoto, Y.,Inoue, H., Nakauchi, H., 1996. In vivo self-renewal of c-Kit+ Sca-1+ Lin(low/−) hemopoietic stem cells. J. Immunol. 156, 3207.

Pearce, D.J., Ridler, C.M., Simpson, C., Bonnet, D., 2004.Multiparameter analysis of murine bone marrow side populationcells. Blood 103, 2541.

Petersen, T.W., Ibrahim, S.F., Diercks, A.H., van den Engh, G., 2004.Chromatic shifts in the fluorescence emitted by murine thymocytesstained with Hoechst 33342. Cytometry A 60, 173.

Sarkadi, B., Ozvegy-Laczka, C., Nemet, K., Varadi, A., 2004.ABCG2-a transporter for all seasons. FEBS Lett. 567, 116.

Scharenberg, C.W., Harkey, M.A., Torok-Storb, B., 2002. TheABCG2 transporter is an efficient Hoechst 33342 efflux pumpand is preferentially expressed by immature human hematopoieticprogenitors. Blood 99, 507.

Shapiro, H.M., 2003. Practical Flow Cytometry. John Wiley and Sons,Hoboken, New Jersey.

Shapiro, H.M., Perlmutter, N.G., 2001. Violet laser diodes as lightsources for cytometry. Cytometry 44, 133.

Smith, P.J., Nakeff, A., Watson, J.V., 1985. Flow-cytometric detectionof changes in the fluorescence emission spectrum of a vital DNA-specific dye in human tumour cells. Exp. Cell Res. 159, 37.

Steen, H.B., Stokke, T., 1986. Fluorescence spectra of cells stainedwith a DNA specific dye, measured by flow cytometry. Cytometry7, 104.

Stokke, T., Holte, H., Davies, C.D., Steen, H.B., Lie, S.O., 1990.Quenching of Hoechst 33258 fluorescence in erythroid precursors.Cytometry 11, 686.

Szabo Jr., G., Kiss, A., Damjanovich, S., 1981. Flow cytometricanalysis of the uptake of Hoechst 33342 dye by humanlymphocytes. Cytometry 2, 20.

Telford, W.G., Frolova, E.G., 2004. Discrimination of the Hoechst sidepopulation in mouse bone marrow with violet and near-ultravioletlaser diodes. Cytometry 57A, 45.

Terunuma, A., Jackson, K.L., Kapoor, V., Telford, W.G., Vogel, J.C.,2003. Side population keratinocytes resembling bone marrow sidepopulation stem cells are distinct from label-retaining keratinocytestem cells. J. Invest. Dermatol. 121, 1095.

Wolf, N.S., Kone, A., Priestley, G.V., Bartelmez, S.H., 1993. In vivoand in vitro characterization of long-term repopulating primitivehematopoietic cells isolated by sequential Hoechst 33342-rhodamine 123 FACS selection. Exp. Hematol. 21, 614.

Wulf, G.G., Luo, K.L., Jackson, K.A., Brenner, M.K., Goodell, M.A.,2003. Cells of the hepatic side population contribute to liverregeneration and can be replenished with bone marrow stem cells.Haematologica 88, 368.

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., Sorrentino, B.P., 2001. The ABC transporter Bcrp1/ABCG2 isexpressed in a wide variety of stem cells and is a moleculardeterminant of the side-population phenotype. Nat. Med. 7, 1028.