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First published online August 3, 2006
Stem Cells Vol. 24 No. 11 November 2006, pp. 2573 -2581
doi:10.1634/stemcells.2006-0266; www.StemCells.com
© 2006 AlphaMed Press

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TECHNOLOGY DEVELOPMENT

The Minimal Instrumentation Requirements for Hoechst Side Population Analysis: Stem Cell Analysis on Low-Cost Flow Cytometry Platforms

Raquel Cabanaa, Ella G. Frolovab, Veena Kapoorc, Richard A. Thomasa, Awtar Krishand, William G. Telfordc

aNPE Systems, Pembroke Pines, Florida, USA;
bMetabolism Branch,
cExperimental Transplantation and Immunology Branch, Center for Cancer Research, National Cancer Institute-NIH, Bethesda, Maryland, USA;
dUniversity of Miami School of Medicine, Miami, Florida, USA

Key Words. Hoechst side population • Stem cell • Solid state laser • Laser diode • Mercury arc lamp • Light-emitting diode

Correspondence: William G. Telford, Ph.D., National Cancer Institute, Bldg. 10, Rm. 3-3297, 9000 Rockville Pike, Bethesda, Maryland 20892, USA. Telephone: 301-435-6379; Fax: 301-480-4354; e-mail: telfordw{at}mail.nih.gov

Received May 1, 2006; accepted for publication July 14, 2006.
First published online in STEM CELLS EXPRESS   August 3, 2006.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
The Hoechst side population (SP) technique is a critical method of identifying stem cells and early progenitors in rodent, nonhuman primate, and human hematopoietic and nonhematopoietic tissues. In this technique, the cell-permeable DNA-binding dye Hoechst 33342 is loaded into the cell population of interest; stem cells and early progenitors subsequently pump this dye out via an ATP-binding cassette membrane pump-dependent mechanism, resulting in a low-fluorescence "tail" (the SP) when the cells are analyzed by flow cytometry. This population contains stem cells and early progenitors. One significant drawback of this method is the requirement of an UV laser to excite the Hoechst 33342. Unfortunately, flow cytometers equipped with UV sources are expensive to own and operate and are not readily available to many laboratories or institutions. In the interests of designing a less expensive flow cytometric system for stem cell analysis, we determined the minimum UV excitation and instrumentation requirements for measuring Hoechst SP. Less than 3 mW of UV laser output was required for adequate resolution of Hoechst SP on two cuvette-based flow cytometers, one of which was a simple, inexpensive benchtop analyzer (the Quanta Analyzer; NPE Systems). Furthermore, Hoechst SP could also be adequately resolved on this epifluorescence-based cytometer platform using two nonlaser UV sources, a mercury arc lamp with a UV bandpass filter and a UV-emitting light-emitting diode. These results suggest that an economical flow cytometric system can be designed that is capable of resolving Hoechst SP, with a cost far lower than most UV laser-equipped commercial systems. An inexpensive system of this type would make Hoechst SP analysis available to a much broader group of stem cell investigators.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
The Hoechst side population (SP) technique is an important methodology for identifying stem cells and early progenitors in rodent, nonhuman primate, and human hematopoietic and nonhematopoietic tissues [1–4]. In this method, the cell-permeable DNA-binding dye Hoechst 33342 is passively loaded into cells. Stem cells and early progenitors subsequently pump this dye out via an ABC membrane pump-dependent mechanism, whereas more differentiated cells retain the dye. The cells are then analyzed on a flow cytometer equipped with an UV laser, and the Hoechst dye fluorescence is measured through blue (~450 nm) and red (~650 nm) bandpass optics. When Hoechst blue and red fluorescence signals collected and plotted against one another, a low-fluorescence "tail" of cells is observed, relative to the much larger bulk of Hoechst-bright cells. This "tail" has been referred to as the SP [1]. The mechanism of Hoechst SP has been linked to the ABC transporter protein Bcrp1/ABCG2, which effluxes Hoechst 33342 in both rodent and human stem cells [5–9]. When physically separated by fluorescence-activated cell sorting, murine SP cells can reconstitute irradiated and immunodeficient recipient mice at very low frequencies [1, 4]. In vitro stem cell culture assays and simultaneous fluorescent immunophenotyping have further confirmed that the SP region contains stem cells and early progenitors in a variety of mammalian species (including humans and non-human primates) [4].

The simplicity of this assay and its apparently broad applicability across species and tissues has made it a critical technique in stem cell biology. Nevertheless, its requirement for a UV laser source has made it relatively inaccessible to most laboratories. Flow cytometers with UV lasers are not common; traditionally, UV laser light has only been available from large-frame ion lasers such as argon or krypton sources, which are expensive and maintenance-intensive. Large-cell sorters such as the BD Biosciences FACSVantage (BD Biosciences, San Diego, http://www.bdbiosciences.com), the DakoCytomation MoFlo (DakoCytomation, Glostrup, Denmark, http://www.dakocytomation.com), and the Beckman Coulter Altra (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com) are the only instruments that can accommodate such physically large lasers, requiring a significant investment in equipment and infrastructure to detect Hoechst SP. UV laser light also requires expensive instrument optics that are not usually normally incorporated into affordable instruments. Since cytometers that can accommodate UV excitation are generally available only at large institutions, access to this technique by many stem cell researchers is very limited.

Fortunately, recent UV laser technology has provided several alternatives to traditional ion sources. Diode-pumped solid state (DPSS) UV lasers are now available that emit at 355 nm; these lasers can be more expensive than their gas counterparts but are smaller and can be accommodated by benchtop flow cytometer systems [10]. Hoechst SP can be readily detected using these sources [11]. Laser diodes are another recent laser technology that has provided numerous excitation wavelengths at relatively low cost [12–15]. The recent development of blue and violet laser diodes has driven the further development of laser diodes approaching the UV [13–15]. Near-UV laser diodes (NUVLDs) emit a relatively long-wavelength UV beam (365–385 nm) but are good flow cytometer excitation sources and give excellent excitation of Hoechst SP on cuvette-based flow cytometers [10, 11, 16]. This second class of laser is much less expensive than either ion or DPSS sources and has low maintenance demands.

Another important technological development that may contribute to more accessible Hoechst SP detection is the proliferation of small, inexpensive flow cytometers. The Guava Technologies platforms (Guava Technologies, Hayward, CA, http://www.guavatechnologies.com) and the NPE Systems Quanta Analyzer (NPE Systems, Pembroke Pines, FL, http://www.npesystems.com) are only two examples of small cytometers with a single excitation source (laser or lamp), capable of two- or three-color analysis [17]. Although less capable than larger systems, they can be configured for specific applications at very low cost, especially when using inexpensive laser sources. In this study, we have combined a low-cost instrument platform (the NPE Quanta Analyzer) with a variety of inexpensive low-power UV laser sources for the analysis of Hoechst SP. We have also used two nonlaser UV sources for Hoechst SP analysis, namely a mercury arc lamp (which possesses significant UV emission) and a novel UV-emitting light-emitting diode source. All of the laser and nonlaser UV sources tested proved adequate for Hoechst SP analysis on this basic instrument platform. This study demonstrated that Hoechst SP analysis can be carried out on a simple instrument platform at a far lower cost than that of traditional methods of stem cell enumeration.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Cells
Male BALB/c mice from 8 to 20 weeks old were obtained from the National Cancer Institute Laboratory Animal Facility. Mice were euthanized by gradual CO2 exposure according to NIH guidelines. Bone marrow was extracted from femurs and tibias and used with no further fractionation. Frozen human bone marrow and cord blood were obtained from AllCells, LLC (Emeryville, CA, http://www.allcells.com) and thawed immediately prior to use.

Hoechst SP Labeling
Mouse and human hematopoietic cells were labeled with Hoechst 33342 according to the method of Goodell et al. [1] and Goodell [3] with minimal modification. Briefly, cells were suspended in room-temperature Hanks' balanced saline solution containing 2% fetal bovine serum and 2 mM HEPES buffer (SP buffer) at 5 x 106 cells per milliliter. Cells were prewarmed to 37°C, and Hoechst 33342 added to a final concentration of 5 µg/ml, from 1 mg/ml stock in double-distilled H2O. Cells were incubated for 90 minutes, centrifuged, and resuspended in cold SP buffer at 5 x 106 per milliliter. Cells were then kept on ice and analyzed within 6 hours of labeling. Propidium iodide was added to the cells at 2 µg/ml immediately prior to analysis. For some experiments, cells were preincubated with the ABCG2 inhibitor fumetrimorgin C at 37°C for 30 minutes prior to Hoechst 33342 addition.

Flow Cytometry
Hoechst 33342-labeled cells were subsequently analyzed on one of two cuvette-based flow cytometers: [1] a BD LSR II (BD Biosciences), or [2] an NPE Quanta Analyzer (NPE Systems). Each cytometer was systematically equipped with one of several near-UV or UV lasers with descending power levels. These included the following:

  1. An Nd:YVO4 solid state UV laser. Emission was at 355 nm, and power level was 22 mW (Uniphase Lightwave; JDSU, Milpitas, CA, http://www.jdsu.com). This high-frequency pulsed laser source (>50 MHz) functioned as a quasi-continuous wave source. This more powerful laser was used as a positive performance baseline.
  2. A dual-module NUVLD. This unit combines the polarized beams from two NUVLD lasers into a single beam, using a polarized beam splitter cube. Emission was at 375 nm, and power level was 18 mW (Power Technology, Alexander, AR, http://www.powertechnology.com).
  3. A single-module NUVLD. Emission was at 374 nm, and power level was 8 mW (Coherent, Mountain View, CA, http://www.cohr.com; Power Technology).
  4. A single NUVLD coupled to single-mode fiber optic, with collimation optics. Emission was at 374 nm, and power level postfiber was approximately 2.3 mW (Point Source Ltd., Southampton, U.K., http://www.point-source.com).

Many of these lasers are described in more detail in ref. 10.

The NPE Quanta used an epifluorescence optical path and could be equipped with two nonlaser UV sources, both with condenser optics:

  1. A mercury arc lamp with a 365 nm bandpass excitation filter. Total lamp power was 80 W. A 365 nm bandpass excitation filter with infrared blocking was used to isolate the UV lamp emission. Mercury arc lamps as excitation sources are discussed in refs. 17 and 18.
  2. A UV-emitting light-emitting diode (iLED, Nichia, Japan, http://www.nichia.com). Emission wavelength was 365 nm, and total power was 100 mW. The UV light-emitting diode (LED) system, heat sink, and diode drivers were designed and built by Power Technology. LEDs as light sources for cytometry are discussed in 19.

The Hoechst SP blue and red fluorescence signals were detected on the BD LSR using a 450/50 nm bandpass and a 650 nm longpass filter, respectively, with a 580 nm longpass reflecting dichroic to separate the signals. The Hoechst SP blue and red fluorescence signals were also detected on the NPE Quanta using the same 450/50 nm bandpass and a 650 nm longpass filter, respectively, but with a 610 shortpass reflecting dichroic for signal separation. On the BD LSR II, propidium iodide fluorescence was detected using a 488 nm primary laser source, through a 585/26 nm bandpass filter. On the NPE Quanta, propidium iodide was excited using the UV source and detected through the 650 nm longpass dichroic. Data were acquired with FACSDiVa version 4.0 acquisition software (for the BD LSR II) or CellQuanta version 1.0 (for the NPE Quanta). Postacquisition data analysis and display was done using FlowJo version 5.7 for Windows (Tree Star Software, Ashland, OR). Instrument alignments were carried out for both laser and nonlaser sources with InSpeck Blue UV-excited microsphere arrays (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com), using both bright and dim microspheres to optimize instrument alignment. InSpeck Blue arrays were similarly used to compare instrument/laser sensitivity and fluorescence resolution.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 Acknowledgments
 References
 
Hoechst SP analysis was tested on two instrument platforms using a variety of UV sources. The BD LSR II is a standard multilaser analysis platform with a quartz cuvette flow cell with orthogonal collection optics with a numerical aperture of 1.2. This light-sensitive system has enabled the use of low-power laser sources for the detection of very low fluorescence signals, including Hoechst SP [11]. Its open-architecture design also allows the installation of a number of different laser sources. In this study, a variety of near-UV and UV lasers ranging from more than 20 mW to less than 3 mW were mounted on this system and assessed for their ability to adequately detect Hoechst SP. The second system, an NPE Analyzer, also used an enclosed flow cell. However, it also used an epifluorescence optical path, with an objective numerical aperture similar to that of the BD LSR II. This epifluorescence system allowed the use of both the same laser systems as above and two nonlaser UV sources, a mercury arc lamp, and a UV-emitting high-intensity LED. These two instrument systems allowed the evaluation of a broad range of laser wavelengths and power levels for Hoechst SP analysis, including two nonlaser sources.

The BD LSR II system is shown in Figure 1. The default configuration is shown in Figure 1A, with a Lightwave Nd:YVO4 frequency-tripled UV laser source. This laser emits a 355 nm UV line at more than 20 mW and is a typical laser source for Hoechst SP analysis. An NUVLD is shown in Figure 1B. This source emits at a slightly longer 375 nm and normally does not exceed 10 mW in power. Both the Nd:YVO4 and NUVLD sources have been previously shown to be good excitation sources on the LSR II using fluorescent standards [10], and NUVLDs have been shown to give Hoechst SP resolution with mouse bone marrow [16]. Figure 1C shows an even lower-power configuration, an NUVLD coupled to a single-mode fiber optic. Single-mode fibers allow beam transmission while maintaining beam profile and polarization characteristics, and they are frequently used in benchtop cytometry systems, which benefit from the flexibility this type of beam delivery provides. However, near-UV transmission with these fibers is relatively inefficient; in the illustrated system, postfiber beam output is approximately 2.8 mW.


Figure 1
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  		Figure 1. The BD LSR II equipped with several UV and near-UV laser sources. (A): An Nd:YVO4 frequency-tripled solid state UV laser emitting at 355 nm (22 mW); (B): An NUVLD emitting at 374 nm (8 mW); (C): A single-mode fiber-coupled NUVLD emitting at 375 nm (10 mW prefiber, 2.3 mW postfiber). Abbreviation: NUVLD, near-UV laser diode.

 
Nevertheless, even this low level of UV emission was sufficient to resolve Hoechst SP in mouse bone marrow. The top row of histograms in Figure 2 illustrates the analysis of an InSpeck Blue microsphere array using all of the above laser systems on the LSR II. This array consists of multiple microsphere populations with decreasing fluorescence, starting from an arbitrary 100% and descending to 30%, 10%, 3%, 1%, 0.3%, and unlabeled. Instrument sensitivity can be assessed by the ability of a particular laser/detector/filter configuration to resolve the dimmest bead populations from one another and from the unlabeled population. Figure 2 illustrates that the dimmest bead population (0.3%) could be resolved from unlabeled beads with both the most powerful and least powerful laser sources. Although more powerful UV sources have been previously shown to give better low-fluorescence detection sensitivity than less powerful sources [10], the fiber-coupled source illustrated here still gave a degree of instrument sensitivity not significantly different from more powerful sources (with small differences in their fluorescence sensitivity only reflected in increased peak CV of the dim microsphere population as the laser power decreased). In the bottom row of histograms in Figure 2, analysis of Hoechst SP indeed showed adequate SP resolution using the 2.8-mW UV laser source, not significantly different in appearance from analysis with the 22-mW source. The G0/G1 peak did expand somewhat at the lowest laser power, but this effect did not appear to interfere with SP resolution. This extremely low level of UV excitation was therefore sufficient to visualize Hoechst SP on this cuvette-based flow cytometry platform.


Figure 2
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  		Figure 2. Hoechst SP analysis on the BD LSR II using UV laser sources with descending power levels. Top row, InSpeck Blue microsphere array analysis using an Nd:YVO4 laser emitting at 355 nm (22 mW) (left histogram), an NUVLD emitting at 374 nm (8 mW) (middle histogram), or a fiber-coupled NUVLD emitting at 375 nm (2.3 mW) (right histogram). Bottom row, Hoechst SP analysis of mouse bone marrow using an Nd:YVO4 laser emitting at 355 nm (22 mW) (left histogram), an NUVLD emitting at 374 nm (8 mW) (middle histogram) or a fiber-coupled NUVLD emitting at 375 nm (2.3 mW) (right histogram). CVs of the 0.3% microsphere population are indicated in parentheses. Abbreviation: NUVLD, near-UV laser diode.

 
The second evaluated platform was an NPE Quanta Analyzer (Fig. 3A). The NPE Quanta also uses a cuvette flow cell, but of a different design than the LSR II. Unlike the orthogonal geometry of the LSR II flow cell and objective, the NPE Quanta uses an epifluorescence optical path, where both the excitation source and the emission signals are transmitted via the same objective. The flow cell consisted of a 125-µm capillary, bounded on one side by a thin glass wall (shown in Fig. 3B mounted on an x-y stage). This design allows the light collecting objective to be placed in very close proximity to the capillary with immersion oil coupling (similar to the optical gel interface on the LSR II cuvette), allowing similar light transmission efficiency. The epifluorescence design also allows both laser and nonlaser sources to be configured into the instrument.


Figure 3
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  		Figure 3. NPE Systems Quanta Analyzer. (A): Exterior of the unit. (B): Alignment stage and direct impedance flow cell. The excitation objective is located below the flow cell and is not visible. Laser and nonlaser sources were aligned in the direction indicated by the arrow below the stage and reflected up through the objective and the flow cell.

 
As with the LSR II, several UV laser sources were mounted on the NPE Quanta. The most powerful was a dual-module NUVLD system. This system combined the beams of two polarized NUVLDs (10 mW each) into a single beam path with a total power level of more than 18 mW. The resulting dual beam was indistinguishable from a single beam and could be aligned to the instrument as would a normal laser. A single-module NUVLD emitting at 8 mW and the fiber-coupled 2.3-mW unit illustrated above were also tested (Fig. 4A–4C). InSpeck Blue microsphere array analysis again confirmed that sensitivity and resolution in the range necessary for Hoechst SP resolution was adequate and similar for the full range of laser sources (Fig. 4, bottom histogram row). Similarly, good Hoechst SP resolution was obtainable even from the 2.8-mW source (Fig. 4, bottom histogram row). Again, the G0/G1 peak expanded somewhat but did not appear to interfere with SP resolution.


Figure 4
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  		Figure 4. Hoechst SP analysis on the Quanta with UV laser sources with descending power levels. (A): Instrument interior, alignment stage, and flow cell, with an NUVLD emitting at 374 nm (8 mW) (circled) mounted and aligned to the flow cell. (B): A fiber-coupled NUVLD emitting at 375 nm (2.3 mW) on the Quanta (circled). (C): The fiber output aligned to the stage and flow cell (circled). Top row, InSpeck Blue microsphere array analysis using a dual-module NUVLD emitting at 374 nm (18 mW) (left histogram), a single-module NUVLD emitting at 374 nm (8 mW) (middle histogram), and a fiber-coupled NUVLD emitting at 375 nm (2.3 mW) (right histogram). Bottom row, Hoechst SP analysis of mouse bone marrow using a dual-module NUVLD emitting at 374 nm (18 mW) (left histogram), a single-module NUVLD emitting at 374 nm (8 mW) (middle histogram), and a fiber-coupled NUVLD emitting at 375 nm (2.3 mW) (right histogram). CVs of the 0.3% microsphere population are indicated in parentheses. Abbreviation: NUVLD, near-UV laser diode.

 
Finally, the NPE Quanta was configured with two nonlaser UV sources, a mercury arc lamp and a UV-emitting LED. Noncoherent light sources have been integrated into flow cytometers since their original development, but they present special challenges in directing their light output to the cells. For both sources, asymmetric condenser optics are required to focus sufficient amounts of light to the instrument cuvette, similar to those found on epifluorescence microscopy systems; even in the best-case scenario, transmission is not particularly efficient, and large amounts of light are lost. Nevertheless, both arc lamps and high-intensity LEDs provide quiet, stable UV excitation; mercury lamps have historically been used for flow cytometric DNA content analysis because of these characteristics [18]. They are also extremely economical sources of high-intensity light (measured in the hundreds rather than tens of thousands of dollars), making them of great interest when designing inexpensive cytometry systems requiring UV excitation.

The mercury arc lamp excitation for the NPE Quanta is shown in Figure 5A–5C. The lamp was contained within a protective housing, with a condenser lens and UV excitation filter (365 nm) mounted in front. The entire unit was mounted on the NPE Quanta, and both the lamp and stage aligned to the UV emission path. InSpeck Blue microsphere analysis is shown in the histograms in Figure 5: a single 100% bead population on linear scale (top) and a full array (bottom). The full array down to 0.3% was distinguishable from the unlabeled population, although resolution was less than that observed even with the lowest-power laser sources shown above.


Figure 5
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  		Figure 5. NPE Quanta equipped with a mercury arc lamp. (A, B): Hg arc lamp assembly, with condenser optics and with a 365 nm bandpass excitation filter in place. (C): The lamp housing in place and aligned to the stage and flow cell. Top histogram, InSpeck Blue 100% microsphere analysis with arc lamp excitation, with peak CV and detector gain indicated. Bottom histogram, InSpeck Blue microsphere array analysis with arc lamp excitation.

 
The second nonlaser source evaluated was a UV-emitting LED. High-intensity LEDs are attracting attention as excitation sources for many fluorescence detection systems. The Nichia iLEDs illustrated in Figure 6 emit at 365 nm, with an average power level of 100 mW. Five of them are assembled in an array in Figure 6A. A magnified examination of one LED in Figure 6B shows their extremely small surface area; when their focused emission is projected onto a surface, their structure of 18 distinct dye subunits is visible. The emission of one such grid was focused using an asymmetric condenser element shown in Figure 6D and mounted on the NPE Quanta in place of the mercury lamp (Fig. 6E). Analysis of InSpeck Blue 100% microspheres (Fig. 6, top histogram) showed that the population CV and required detector gain increased compared with the mercury arc lamp, indicating that the LED was providing less excitation. Similarly, only the 1% InSpeck Blue population was distinguishable from the unlabeled background when the entire microsphere array was analyzed (Fig. 6, bottom histogram).


Figure 6
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  		Figure 6. NPE Quanta equipped with UV LED. (A): An array of Nichia iLED UV-emitting LEDs, mounted on a heat sink with integrated diode driver electronics. (B): Close-up of one diode grid. (C): Focused image of single-diode grid (18 distinct dyes). (D): UV LED mounting assembly with condenser optics. (E): UV LED assembly mounted on NPE Quanta, and aligned to the stage and flow cell. Top histogram, InSpeck Blue 100% microsphere analysis with single-UV LED grid excitation, with peak CV and detector gain indicated. Bottom histogram, InSpeck Blue microsphere array analysis with a single-UV LED grid excitation. Abbreviation: LED, light-emitting diode.

 
Nevertheless, despite the reduced instrument sensitivity with these nonlaser sources, they still gave acceptable Hoechst SP resolution. Figure 7 shows analysis of mouse bone marrow, human bone marrow, and human cord blood using the mercury lamp (left column) or the UV LED (middle column), in comparison with the conventional solid-state UV laser on the LSR II (right column). In all cases, a tightly defined SP could be resolved. Propidium iodide (PI) as a viability marker was particularly critical with these sources, since apoptotic cells tended to occlude the SP region (data not shown). The PI-positive cells were visible in the Hoechst red bright region of the scatter plots, but were well separated from the SP region.


Figure 7
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  		Figure 7. Hoechst SP analysis on the NPE Quanta using mercury arc lamp and UV LED excitation sources. Left column, Hoechst SP analysis of mouse bone marrow (top histogram), human bone marrow (middle histogram), and human cord blood (bottom histogram) using mercury arc lamp excitation. Middle column, Hoechst SP analysis of mouse bone marrow (top), human bone marrow (middle), and human cord blood (bottom) using UV LED excitation. Right column, Hoechst SP analysis of mouse bone marrow (top), human bone marrow (middle), and human cord blood (bottom) on a BD LSR II using Nd:YVO4 laser excitation (355 nm, 22 mW).

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 Acknowledgments
 References
 
Hoechst SP is a very valuable technique for stem cell characterization. Nevertheless, access to this technique has been limited by the limited availability and prohibitive cost of UV laser sources on flow cytometers. The flow cytometer platforms capable of accommodating UV lasers have also been rather limited, requiring either large frame cell sorters that can accommodate large argon or krypton ion lasers, or polychromatic flow cytometers equipped with UV-compatible optics and solid state laser sources. Both of these systems are prohibitively expensive for many institutions. We have previously demonstrated that low-cost NUVLDs are capable of analyzing Hoechst SP. In this study, we have determined that the minimum threshold of UV excitation required for Hoechst SP analysis is very low (less than 3 mW), allowing the use of both low-power UV sources and fiber-coupled NUVLDs for this application. These laser types could also be implemented on inexpensive flow cytometer platforms like the NPE Quanta, a system that is one-fifth the cost of the typical multilaser cytometer. In addition, the epifluo rescence-based cytometer could also accommodate nonlaser sources. Although it is extremely difficult to model and measure, the effective amount of UV light reaching the cell intercept region on the NPE Quanta is probably no more than 1–2 mW, further illustrating the amount of UV excitation minimally required to detect this phenomenon.

Of course, there are a number of practical problems with this approach that need to be addressed. Both systems used in this study were cuvette-based, with high numerical aperture collection optics closely mated to the cell stream cuvette using a refractive medium (optical gel or immersion oil). Many electrostatic cell sorting systems still rely on jet-in-air cell delivery, where hydrodynamically focused cells are ejected from a nozzle into the laser path. Droplet formation and charging for cell separation require a large amount of space between the cell stream and the collection optics, significantly decreasing the signal collection efficiency. High-power UV lasers are still required in such systems to overcome this limitation. However, more modern cell sorters combine quartz cuvette flow cells and downstream jet-in-air droplet formation and electrostatic separation, eliminating the need for more powerful UV lasers. Lower-power sources should be compatible with this type of cell sorter.

The second issue is the need for polychromatic analysis capability in Hoechst SP analysis. The detection of side population alone is now rarely sufficient for good stem cell enumeration. At a minimum, simultaneous exclusion of lineage-positive cells is usually required, using a series of lineage antibodies (for mature T cells, B cells, myeloid cells, etc.) labeled with a single fluorochrome. The lineage-positive cells can then be excluded by gating, restricting SP analysis to the lineage-negative fraction. Further simultaneous analysis of stem cell- and progenitor-specific markers, such a Sca-1 and c-kit in mouse and CD34 in human, is also necessary for true multiparametric stem cell enumeration. Such multicolor analysis (which can easily exceed five or more fluorescent parameters) is possible on multilaser cytometers such as the LSRII but is still limited on less expensive cytometers, such as the NPE Quanta. It should nevertheless be possible to expand the multicolor capability of small cytometers to accommodate truly informative stem cell analysis without an excessive increase in cost. The cost of producing UV excitation no longer needs to be a prohibitive factor in the design of such instruments, placing stem cell analysis within reach of a much larger group of investigators.


   DISCLOSURES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
References
 
We acknowledge Ernest Thomas, Michael Brochu, Jr., and Michael Brochu, Sr., for instrumentation support and valuable discussion. Fumetrimorgin C was provided by Susan Bates and Robert Robey (Developmental Therapeutics Branch, National Cancer Institute-NIH). This study was supported by intramural research funds provided by the Center for Cancer Research of the National Cancer Institute, National Institutes of Health. E.G.F. is currently affiliated with the Department of Immunology, The Cleveland Clinic, Cleveland, OH. R.C. and E.G.F. contributed equally to this work.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 

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