showcase.par.79408.image.0.0.1

In this study, we demonstrate the ability of the Attune® Acoustic Focusing Cytometer with the blue/ violet laser configuration to quickly analyze a large number of events in search of very rare populations of stem cells. The Attune® Acoustic Focusing Cytometer is equipped with a violet laser light source and is able to analyze cells at flow rates up to 1,000 µL/min, making it the ideal platform for side population analysis.

Learn more about the Attune® Cytometer

Download the Stem Cells app note

Introduction

Stem and progenitor cells have now been identified in tissues throughout the body. A technique known as side population analysis can be used to distinguish progenitor cells from somatic cells based on the activity of ATP-Binding Cassette pumps. These pumps are found in the plasma membrane and specifically efflux environmental toxins and foreign molecules out of cells [1,2]. One of these pumps—the ABCG2 efflux pump—is active in many types of stem cells and has been shown to pump the cell-permeant DNA-binding dye Vybrant® DyeCycle™ Violet out of stem cells [3]. This effect can be used in flow cytometry to identify stem cells based on the differential efflux of dye: differentiated cells retain the dye inside, where it selectively labels DNA and results in a bright fluorescent signal, while stem cells and progenitor cells actively pump the dye out. When dye emission is analyzed on a flow cytometer equipped with a 405 nm laser in a two-parameter display of red and blue emission wavelengths, a tailing population exhibiting dim fluorescence (representing the stem cell population) is observed relative to the majority of cells with bright fluorescence. This tail is called the side population. This efflux activity of stem cells can be confirmed by addition of compounds such as fumitremorgin C which inhibit the action of the ABCG2 pump, providing a valuable control to confirm dye efflux and the probable presence of stem cells (Figure 1).

As mentioned above, in this study we demonstrate the ability of the Attune® Acoustic Focusing Cytometer with the blue/ violet laser configuration to quickly analyze a large number of events in search of very rare populations of stem cells. The Attune® Acoustic Focusing Cytometer is equipped with a violet laser light source and is able to analyze cells at flow rates up to 1,000 µL/min, making it the ideal platform for side population analysis.  Vybrant® DyeCycle™ Violet is used in single color to demonstrate the side population technique in cultured human adenocarcinoma and primary human corneal epithelial cells. Guidelines are also provided for designing and performing multiplexed experiments incorporating immunophenotyping with Vybrant® DyeCycle™ Violet stain to confirm identity of rare hematopoietic stem cells (HSC) in normal mouse and human bone marrow samples.

Hematopoietic Stem Cell Identification in Human Bone Marrow

Hematopoietic stem cells (HSCs) are a small subset of bone marrow cells that can regenerate the entire hematopoietic system when transplanted into a recipient. Complex immunophenotyping experiments [3] have demonstrated that human HSCs can be defined as positive for CD34, negative for CD38, and negative for markers of differentiated hematopoietic cell types. Here we used a premixed cocktail of FITC-labeled mouse anti-human antibodies (CD2, CD3, CD14, CD16, CD19, CD56, and CD235a) to identify differentiated cells in a single channel. The cells of interest in this experiment—precursor and stem cells—will not be labeled with this cocktail. The viability dye LIVE/ DEAD® Fixable Green Dead Cell Stain is used to very brightly stain dead cells in this experiment so that they can be removed from the analysis. Both the lineage cocktail and viability dye are detected in the BL1 channel of the Attune® Acoustic Focusing Cytometer, so that a gate on the negative population of cells will contain only live precursor and stem cells—dead cells and differentiated cell types (positive population of cells) are excluded from the analysis. The HSC subpopulation is typically 0.05% to 0.10% of bone marrow cells, requiring the analysis of a large number of cells to generate statistically significant data [4]. The extremely precise sample alignment provided by the Attune® Acoustic Focusing Cytometer enables very high throughput without loss of data quality or integrity. The following procedures will guide you through the process of cell preparation, instrument setup, cell labeling, and data collection.



Figure 1. Vybrant® DyeCycle™ Violet dye in human adenocarcinoma cells. Like many types of stem cells, cultured human adenocarcinoma (A549) cells constitutively express the ABCG2 membrane pump. In this example, A549 cells are used to verify action and inhibition of ABCG2. (A) Staining of A549 cells for 90 min at 37°C with 5 µM Vybrant® DyeCycle™ Violet results in a poor DNA content histogram because the dye is actively pumped out of cells by the ABCG2 membrane pump. (B) Treatment of A549 cells with the ABCG2 inhibitor fumitremorgin C (10 µM) results in a typical DNA content histogram indicating retention of stain in the cells. (C, D) Vybrant® DyeCycle™ Violet has a broad fluorescence emission which can be detected in the VL1 and VL3 channels (450/50 and 603/48 nm bandpass filters, respectively) of the Attune® cytometer with the blue/violet configuration. Dual-parameter plots of VL1 vs. VL3 provide better discrimination between cells of the side population phenotype, those actively effluxing Vybrant® DyeCycle™ Violet Stain and displaying the side population tail (C), and cells treated with the ABCG2 membrane pump inhibitor fumitremorgin C (D). This type of dual-parameter plot will be used to display side population data in the remaining examples of this application note.


Table 1. Staining strategy and single-color and FMO controls for human bone marrow HSC analysis.

    Sample Vybrant®
DyeCycle™ Violet
LIVE/DEAD®
Fixable Green
FITC lineage
cocktail
CD34
R-PE
CD38 TRI-COLOR®
conjugate
Single-color
controls
VL1Bone marrow
    
BL1ArC™ Beads 
   
BL2AbC™ Beads   
 
BL3AbC™ Beads    
FMO
controls
1Bone marrow
  
2Bone marrow
 
3Bone marrow
 
4Bone marrow 
Samples Bone marrow


Materials



Bone marrow cell preparation

  1. Thaw a sample of frozen human bone marrow mononuclear cells in an appropriate medium such as DMEM supplemented with 10% FBS and warmed to 37°C, or according to manufacturer’s directions. Alternatively, fresh bone marrow can be prepared using density gradient separation or by a red blood cell lysis procedure such as ammonium chloride.
  2. Add 200 μg DNase I per milliliter of cells, and incubate for 10 min to reduce cell clumping.
  3. Wash cells once with DMEM + 10% FBS, and resuspend the pellet in DMEM + 10% FBS at approximately 1 x 106 cells/mL.



Cell labeling procedure

  1. Place 1 mL cells (at 1 x 106 cells/ mL in DMEM + 10% FBS) into a 12 x 75 mm tube.
  2. Add 1 μL Vybrant® DyeCycle™ Violet for a 5 μM staining concentration.
  3. Incubate 90 min at 37°C, protected from light.
  4. Wash samples in 3 mL DMEM +10% FBS, centrifuge at 400 x g for 5 min, discard supernatant, and resuspend cells in 1 mL protein-free phosphate-buffered saline (PBS). NOTE: The use of protein-free buffer is required for proper amine-reactive labeling in the next step.
  5. Add 1 μL/mL LIVE/DEAD® Fixable Green Dead Cell Stain.
  6. Incubate 20 min at room temperature, protected from light.
  7. Wash cells: add 3 mL DMEM + 10% FBS to tubes, centrifuge at 400 x g for 5 min, discard supernatant, and resuspend in 100 μL DMEM + 10% FBS.
  8. Use experimentally determined optimal titer or manufacturer’s recommended concentrations to label cells with the mouse anti–human CD34 R-PE and CD38 TRI-COLOR® antibody conjugates, and the FITC-conjugated human lineage cocktail (all three labels are added to the same tube). Incubate 20 min at room temperature, protected from light.
  9. Wash cells: add 3 mL DMEM + 10% FBS to tubes, centrifuge at 400 x g for 5 min, discard supernatant, and resuspend in 1 mL DMEM + 10% FBS for analysis.



Single-color compensation control setup

Spectral overlap (spillover) of the fluorochromes in this experiment must be corrected by determining appropriate
compensation using single-color controls. Some of these controls can be easily prepared with compensation beads, which preserves precious cells. Preparation of the Vybrant® DyeCycle™ Violet cell control takes 90 minutes and should be performed at the same time as cell labeling (previous procedure).

  1. Add one drop of AbC™ capture beads (component B) from the AbC™ Anti-Mouse Bead Kit to each of two microcentrifuge tubes or 12 x 75 mm flow cytometry tubes. Label the beads in one tube with CD34 R-PE and in the other tube with CD38 TRI-COLOR® conjugate. Both antibodies should be used at the experimentally determined optimal titer or manufacturer’s recommended concentration.
  2. Add one drop of ArC™ reactive beads (component A) from the ArC™ Amine Reactive Compensation Bead Kit to one 12 x 75 mm tube, and add 1 μL LIVE/DEAD® Fixable Green Dead Cell Stain.
  3. Incubate bead samples from steps 1 and 2 for 20 min at room temperature, protected from light.
  4. Add 3 mL PBS to bead samples, centrifuge at 400 x g for 5 min, discard supernatant and resuspend in 500 μL PBS.
  5. Add one drop of the appropriate (ArC™ or AbC™) negative bead (component A) to each bead tube.
  6. Add 1 x 106 bone marrow cells in 1 mL of DMEM + 10% FBS to a 12 x 75 mm tube. Add 1 μL Vybrant® DyeCycle™ Violet per milliliter of cells to create a 5 μM staining concentration.
  7. Incubate 90 min at 37°C, protected from light.

    NOTE: A single-color control is not used for the FITC-conjugated lineage cocktail because LIVE/ DEAD® Fixable Green Dead Cell Stain is used as a control for theBL1 channel.



Creating fluorescence-minus-one (FMO) controls

FMO controls are used to set markers appropriately in dual-parameter plots during analysis [7,8].

1. Add approximately 106 cells to each of four 12 x 75 mm tubes.
2. Label cells in each tube according to the Cell labeling procedure above, except omit a different one of the four fluorochromes from the tubes as follows:
- Tube 1: All fluorochromes except LIVE/DEAD® Fixable Green Dead Cell Stain and the FITC-conjugated human lineage cocktail
- Tube 2: All fluorochromes except CD34 R-PE conjugate
- Tube 3: All fluorochromes exceptCD38 TRI-COLOR® conjugate
- Tube 4: All fluorochromes except Vybrant® DyeCycle™ Violet


Setting PMT voltages and compensation

1. Verify instrument performance with Attune® Performance Tracking Beads.
2. Create a new experiment.
3. In the experiment browser, right click on Compensation and select Compensation setup.
4. In the compensation setup dialog box, click to compensate on Area and select parameters for VL1, VL3, BL1, BL2, and BL3.
5. Click OK to create the compensation setup samples.
6. Open the workspace by double-clicking on the first sample in the experiment browser.
7. Create the following plots:
- Fluorescence histograms for VL1-A, BL1-A, BL2-A, and BL3-A
- Density or dot plots for VL1-A vs. VL1-H, VL1-A vs. VL3-A, VL1-A vs. BL1-A, and BL2-A vs. BL3-A
8. Run the four single-color controls to optimize PMT voltage settings without recording to ensure both positive and negative populations are on scale. Voltage thresholds may need to be adjusted to eliminate debris from analysis.
9. Once PMT voltage settings are optimized, double-click the BL1 single-color control tube in the compensation section of the experiment explorer.
10. Forward and Side Scatter voltages may need to be adjusted to place the beads on scale. Do not change the fluorescent PMT settings from those optimized for your cells. Compensation controls and the stained cell panel must be run with the same voltages to obtain a valid compensation matrix.
11. Run the single-color controls for LIVE/DEAD® Fixable Green Dead Cell Stain in BL1, CD34 R-PE conjugate in BL2, and CD38 TRICOLOR® conjugate in BL3. Position the R1 gate around the main population of beads in the dualparameter
FSC vs. SSC plot, and place the R2 marker around the positive peak in the fluorescence histogram for each respective conjugate. Hint: Right-clicking on the R1 gate and selecting Apply gate shape to all controls will keep the gate surrounding the main bead population for all single-color bead controls.
12. Double-click on the first sample in the experiment browser.
13. Run the single-color control for Vybrant® DyeCycle™ Violet, collecting at least 10,000 total events.
14. Use the compensation slider bar in the VL1-A vs. BL1-A density plot to remove VL1 fluorescent spillover from the BL1 channel.


Data collection

1. Set the acquisition volume, collection rate, threshold, and recording criteria as desired. For the data presented here, a 1,000 μL acquisition volume, Standard 200 μL/min collection rate, FSC Threshold of 10,000, and the “record continuously” settings were used.
2. Create four new samples in the Attune® Experiment Browser. Use these to acquire at least 50,000 events for each of the FMO controls.
3. Use the FMO control (minus FITC) to create a histogram gate on the negative population of cells in the BL1 histogram (Figure 2A).
4. Use the FMO control (minus R-PE) in a dual-parameter plot of BL1 vs. BL2 to create a quadrant gate to include the negative cells in BL2 (Figure 2B).
5. Use the FMO control (minus TRI-COLOR® conjugate) in a dualparameter plot of BL1 vs. BL2 to
adjust the quadrant gate to include the negative cells in BL3 (Figure 2C).
6. Use the FMO control (minus Vybrant® DyeCycle™ Violet) to verify that no fluorescent signal appears in a dual-parameter plot of VL1 vs. VL3.
7. Proceed with recording data for test samples. Acquisition of at least one million total events is recommended.




Figure 2. FMO controls for marker placement. (A) The FMO control (minus FITC) is used for marker placement in the BL1 histogram to define the delineation between positive and negative cells in the BL1 channel. (B) The FMO control (minus R-PE) is used to for marker placement to define the delineation between positive and negative cells in the BL2 channel, as seen in the dual-parameter plot of BL1 vs. BL2. (C) The FMO control (minus TRI-COLOR® conjugate) is used for marker placement to define the delineation between positive and negative cells in the BL3 channel as seen in the dual-parameter plot of BL2 vs BL3.




Figure 3. Gating hierarchy to identify HSCs. (A) A dual-parameter plot of Vybrant® DyeCycle™ Violet height vs. area is used to eliminate aggregated cells by creating a region around singlet cells as shown. (B) The singlets defined in plot A are set as the population for histogram plot B; the LIVE/DEAD® Fixable Green Dead Cell Stain and the FITC-conjugated human lineage cocktail are both detected in the BL1 channel. Gating on the negative peak as shown in histogram plot B restricts the analysis to live, undifferentiated cells. (C) The live, lineage-negative (Lin–) cells identified in histogram plot B are used as the population for a dual-parameter plot, CD34 (BL2) vs. CD38 (BL3) which is used to further restrict the analysis to CD34+ CD38– cells. The quadrant markers are placed using FMO controls as defined in Figure 2. (D) The CD34+ CD38– cells from plot C are displayed on a dual-parameter plot of Vybrant® DyeCycle™ Violet red (VL3) vs. blue (VL1) emission. The side population (SP) gated in plot D identifies HSCs based on Vybrant® DyeCycle™ Violet efflux and contains approximately 67% of the Lin– CD34+ CD38– stem cells, or approximately 0.1% of all bone marrow cells. This region was placed on the distinct side population of cells that appears when all live, Lin– events are displayed in this figure. The addition of a duplicate sample in which cells are treated with 5 µM fumitremorgin C to inhibit the ABCG2-mediated dye efflux is useful to confirm the correct placement of this gate.

Back to top

Hematopoietic Stem Cell Identification in Murine Bone Marrow

HSCs can also be identified in mouse bone marrow, using a similar cocktail of antibodies in combination with Vybrant® DyeCycle™ Violet side population analysis. Bone marrow cells are collected from adult mouse femurs, erythrocytes are removed by ammonium chloride lysis, and cells are then treated with DNase I to reduce clumping. The cell staining protocol is very similar to the above procedure used with human bone marrow, with the addition of a second sample in which cells are treated with 5 µM fumitremorgin C during the 90-minute incubation to inhibit the ABCG2-mediated dye efflux, providing a control to confirm the presence of a stem cell population.

As with human bone marrow, a panel of antibodies is used to identify the HSCs. Here we use a mouse lineage cocktail consisting of Alexa Fluor® 488–conjugated antibodies specific for mouse CD3-e, CD11b, CD45R, Ly-6C/G, and TER-119 to identify and gate out all differentiated cell types [4]. Cells are stained with LIVE/DEAD® Fixable Green Dead Cell Stain for 20 minutes, washed with DMEM + 10% FBS, and then labeled for 20 minutes with the mouse lineage cocktail, the rat anti–mouse c-Kit R-PE, and the rat anti–mouse Sca-1 (Ly-6A/E) PE-Cy®5.5. Mouse HSCs are often referred to as the LSK subset, because they are Lin–, Sca-1+, and c-Kit+. A strong correlation has been observed between cells in the LSK subset and those in the Vybrant® DyeCycle™ Violet side population. LSK cells make up a very small percentage of normal mouse bone marrow cells, necessitating the acquisition of a very large number of cells to achieve statistically significant data. The high-resolution data in this experiment represent one million total cells that were acquired in 2.5 minutes on an Attune® Acoustic Focusing Cytometer.


Table 2. Staining strategy and single-color and FMO controls for human bone marrow HSC analysis.

    Sample Vybrant®
DyeCycle™ Violet
LIVE/DEAD®
Fixable Green
FITC lineage
cocktail
CD34
R-PE
CD38 TRI-COLOR®
conjugate
Single-color
controls
VL1Bone marrow
    
BL1ArC™ Beads 
   
BL2AbC™ Beads   
 
BL3AbC™ Beads    
FMO
controls
1Bone marrow
  
2Bone marrow
 
3Bone marrow
 
4Bone marrow 
Samples Bone marrow



Materials

 

Bone marrow preparation

1. Extract bone marrow from mouse femurs according to your specific animal use protocol [6].
2. Disrupt any remaining bone fragments and filter cells through 70 μm nylon mesh.
3. Add 10 mL of freshly prepared ammonium chloride lysis buffer
(prewarmed to 37°C) per 1 mL of cells and incubate 10 min at room temperature.
4. Wash cells: add 10 mL HBSS to tubes, centrifuge at 400 x g for 5 min, discard supernatant, and resuspend in 10 mL of an appropriate medium such as DMEM with GlutaMAX™ and pyruvate supplemented with 10% FBS and warmed to 37°C.
5. Add 100 μg DNase I per mL of cells at 1 x 106 cells/mL and incubate for 10 min at room temperature to reduce cell clumping.
6. Wash cells: add 3 mL DMEM + 10% FBS to tubes, centrifuge at 400 x g for 5 min, discard supernatant, and resuspend in 1 mL DMEM + 10% FBS at approximately 1 x 106 cells/ mL.


Cell labeling procedure

1. Place 1 mL of cells (at 1 x 106 cells/mL in DMEM + 10% FBS) into each of two 12 x 75 mm tubes, labeled “control” and “test”.
2. Add fumitremorgin C to the tube labeled “control” to a final concentration of 5 μM. Incubate 15 min at 37°C.
3. Add 1 μL Vybrant® DyeCycle™ Violet to both tubes for a 5 μM staining concentration.
4. Incubate 90 min at 37°C, protected from light.
5. Wash cells: add 3 mL HBSS to tubes, centrifuge at 400 x g for 5 min, discard supernatant, and resuspend in 1 mL protein-free HBSS. NOTE: The use of protein-free buffer is required for proper amine-reactive labeling in the next step.
6. Add 1 μL/mL LIVE/DEAD® Fixable Green Dead Cell Stain.
7. Incubate 20 min at room temperature, protected from light.
8. Wash cells: add 3 mL DMEM + 10% FBS to tubes, centrifuge at 400 x g for 5 min, discard supernatant, and resuspend in 1 mL DMEM + 10% FBS.
9. Add the rat anti-mouse antibody conjugates c-Kit R-PE, Sca-1 PE-Cy®5.5, and the rat anti-mouse lineage cocktail.
10. Incubate 20 min at room temperature, protected from light.
11. Wash cells: add 3 mL DMEM + 10% FBS to tubes, centrifuge at 400 x g for 5 min, discard supernatant, and resuspend in 1 mL DMEM + 10% FBS for analysis.


Single-color compensation control setup

Spectral overlap between the fluorochromes in this experiment must be subtracted by determining appropriate compensation using single-color controls. Some of these controls can be easily prepared with compensation
beads, which preserves precious cells. Preparation of the cell controls takes 90 minutes and should be performed at the same time as cell labeling (previous procedure).

1. Add one drop of AbC™ capture beads (component A) from the AbC™ Anti-Rat/Hamster Bead Kit to each of two 12 x 75 mm tubes. Label the beads in one tube with c-Kit R-PE and in the other tube with Sca-1 PE-Cy®5.5. Both antibodies should be used at the experimentally determined optimal titers or the manufacturer’s recommended concentrations.
2. Add one drop of ArC™ reactive beads (component A) from the ArC™ Amine Reactive Compensation Bead Kit to another 12 x 75 mm tube, Add 1 μL LIVE/DEAD® Fixable Green Dead Cell Stain.
3. Incubate all bead samples for 20 min at room temperature, protected from light.
4. Add 3 mL DPBS to bead samples, centrifuge at 400 x g for 5 min, discard supernatant, and resuspend in 500 μL DPBS.
5. Add one drop of the appropriate (ArC™ or AbC™) negative bead (component B) to each tube.
6. Add 1 mL of prepared bone marrow cells to a 12 x 75 mm tube. Add 1 μL Vybrant® DyeCycle™ Violet
per mL of cells for a 5 μM staining concentration. Incubate 90 min at 37°C. NOTE: A single-color control is not
used for the Alexa Fluor® 488–conjugated lineage cocktail because LIVE/DEAD® Fixable Green Dead Cell Stain is used as a control for the BL1 channel.


Creating fluorescence-minus-one (FMO) controls


FMO controls are used to set markers appropriately in dual-parameter plots during analysis [7,8].

1. Add approximately one million cells to each of four 12 x 75 mm tubes.
2. Label cells in each tube according to the cell labeling procedure above, except omit a different one of the four fluorochromes from the tubes as follows:
- Tube 1: All fluorochromes except LIVE/DEAD® Fixable Green Dead Cell Stain and the Alexa Fluor® 488–conjugated mouse lineage cocktail
- Tube 2: All fluorochromes except c-Kit R-PE
- Tube 3: All fluorochromes except Sca-1 PE-Cy®5.5
- Tube 4: All fluorochromes except Vybrant® DyeCycle™ Violet
3. Create four new samples in the Attune® Experiment Browser. Use these to acquire 10,000 events from each of the FMO controls.


Setting PMT voltages and compensation

1. Verify instrument performance with Attune® Performance Tracking Beads.
2. Create a new experiment.
3. In the experiment browser, right click on Compensation and select Compensation setup.
4. In the compensation setup dialog box, click to compensate on Area and select parameters for VL1, VL3, BL1, BL2, and BL3.
5. Click OK to create the compensation setup samples.
6. Open the workspace by double-clicking on the first sample in the experiment browser.
7. Create the following plots:
- Fluorescence histograms for VL1-A, BL1-A, BL2-A, and BL3-A
- Density or dot plots for VL1-A vs. VL1-H, VL1-A vs. VL3-A, VL1-A vs. BL1-A, and BL2-A vs. BL3-A
8. Run the four single-color controls to optimize PMT voltage settings without recording to ensure both positive and negative populations are on-scale. Voltage thresholds may need to be adjusted to eliminate debris from analysis.
9. Once PMT voltage settings are optimized, double-click the BL1 single-color control tube in the compensation section of the experiment explorer.
10. Forward and Side Scatter voltages may need to be adjusted to place the beads on scale. Do not change the fluorescent PMT settings from those optimized for your cells. Compensation controls and the stained cell panel must be run with the same voltages to obtain a valid compensation matrix.
11. Run the single-color controls for the Alexa Fluor® 488–conjugated mouse lineage cocktail in BL1, c-Kit R-PE in BL2, and Sca-1 PE-Cy®5.5 in BL3. Position the R1 gate around the main population of beads in the dual-parameter FSC vs. SSC plot, and place the R2 marker around the positive peak in the fluorescence histogram. HINT: Right-clicking on the R1 gate and selecting Apply gate shape to all controls will keep the gate surrounding the main bead population for all single-color bead controls.
12. Double-click on the first sample in the experiment browser.
13. Run the single-color control for Vybrant® DyeCycle™ Violet, collecting at least 10,000 total events.
14. Use the compensation slider bar in the VL1-A vs. BL1-A density plot to remove VL1 fluorescent spillover from the BL1 channel.


Data collection

1. Set the acquisition volume, collection rate, threshold and recording criteria as desired. For the data presented here, a 1,000 μL acquisition volume, Standard 200 μL/min collection rate, FSC threshold of 10,000, and the “record continuously” setting were used.
2. Collect at least 50,000 events for each of the FMO controls.
3. Use the FMO control (minus Alexa Fluor® 488) to create a histogram gate on the negative population of cells in BL1 histogram (Figure 4A).
4. Use the FMO control (minus R-PE) in a dual-parameter plot of BL1 vs.BL2 to create a quadrant gate to include the negative cells in BL2 (Figure 4B).
5. Use the FMO control (minus PE-Cy®5.5) in a dual-parameter plot of BL1 vs.BL2 to adjust the quadrant gate to include the negative cells in BL3 (Figure 4C).
6. Use the FMO control (minus Vybrant® DyeCycle™ Violet) to verify that no fluorescent signal appears in a dual-parameter plot of VL1 vs.VL3 (Figure 4D).
7. Proceed with recording data for samples. Acquisition of at least one million total events is recommended (Figures 5 and 6).


Conclusion

Side population analysis based on ABCG2-mediated efflux of the DNA binding dye Vybrant® DyeCycle™ Violet provides a robust method to identify hematopoietic stem cells in samples of murine bone marrow cells. The side population phenotype correlates well with the common cell surface marker technique of identifying HSCs based on the Lin–/Sca-1+/c-Kit+ phenotype. HSCs comprise a very small fraction of total bone marrow mononuclear cells, so very large event files are required to identify a statistically significant number of stem cells. As shown in this example, the Attune® Acoustic Focusing Cytometer enables acquisition of high-quality data from one million cells in a few minutes.




Figure 4. FMO controls for marker placement. (A) The FMO control (minus Alexa Fluor® 488) is used for marker placement in the BL1 histogram to define the delineation between positive and negative cells in the BL1 channel. (B) The FMO control (minus R-PE) is used to for marker placement to define the delineation between positive and negative cells in the BL2 channel as seen in the dual-parameter plot of BL2 vs. BL3. (C) The FMO control (minus TRI-COLOR® fluorochrome) is used for marker placement to define the delineation between positive and negative cells in the BL3 channel as seen in the dual-parameter plot of BL2 vs. BL3. (D) The FMO control (minus Vybrant® DyeCycle™ Violet) is used to verify that no events are present in the dual-parameter plot of VL1 vs. VL3 and confirms that the other fluorochromes in the experiment do not spill over into these channels.




Figure 5. Identification of HSCs in mouse bone marrow. As with the previous example, a gating strategy is first used to remove aggregated cells from the analysis (not shown), by creating a region around singlet cells for further gating. (A) A histogram gate is placed on the histogram in BL1 (gated on singlet cells) to eliminate dead and differentiated cells from analysis. (B) A dual-parameter plot of Sca-1 vs. c-Kit (BL3 vs. BL2) is then used to gate on the c-Kit+/Sca-1+ population. This population is then displayed on a dual-parameter plot of Vybrant® DyeCycle™ Violet red (VL3) vs. blue (VL1) emission (C). The bone marrow subset containing the most primitive HSCs appears as a narrow side population in this figure. This side population contains approximately 61% of the Lin–/Sca-1+/c-Kit+ stem cells, or approximately 0.1% of all bone marrow cells. (D) Treatment with 5 μM fumitremorgin C inhibits ABCG2-mediated dye efflux out of stem cells, causing a shift in the side population from dim to bright. This control provides valuable evidence that cells in the Vybrant® DyeCycle™ Violet side population are actually stem cells and confirms correct placement of the side population region.




Figure 6. Experimental statistics of the mouse bone marrow subset. The most primitive mouse hematopoietic stem cells defined in this experiment are extremely rare and account for approximately 1 in 1,000 bone marrow cells. These statistics are derived from the data shown in Figure 5C.

Back to top

Human Corneal Progenitor Cell Identification

The Vybrant® DyeCycle™ Violet side population technique can be used to identify stem cells in tissues other than those in the hematopoietic system. For example, a stem cell population which regenerates the corneal surface of the eye can be identified using the side population technique. Here we demonstrate how limbal stem cells (LSCs) can be identified in a population of differentiated corneal cells. For this experiment, Gibco® Human Corneal Epithelial Cells were grown in culture and incubated with 5 μM Vybrant® DyeCycle™ Violet in the presence or absence of 5 μM fumitremorgin C for a total of 90 minutes at 37°C. Figure 7A shows the analysis of 120,000 events on the Attune® Acoustic Focusing Cytometer revealing a very small, distinct side population. This population disappears upon treatment with fumitremorgin C (Figure 7B), confirming that the side population is composed of LSCs.

Materials



Preparation of primary human corneal epithelial cells

1. Grow Gibco® Human Corneal Epithelial Cells in culture conditions in Keratinocyte Serum Free Medium (KSFM) to 70–80% confluency.
2. Decant the medium from the flask or plate. Rinse with 5 mL of DPBS without calcium and without magnesium. Decant the DPBS.
3. Add an appropriate volume (e.g., 2 mL in a 75 cm2 flask) of prewarmed TrypLE™ Select reagent. Rock the vessel to coat the cell sheet completely.
4. Incubate at 37°C until the cells have detached (observe at 5-minute intervals.) Gently tap the vessel to dislodge the cells.
5. Dilute in 2 to 5 mL of KSFM and transfer the cell suspension to a 15 mL conical centrifuge tube. Centrifuge for 5 to 10 min at 100 x g. Discard the supernatant and suspend the cell pellet in 2 to 5 mL of fresh KSFM at approximately 1 x 106 cells/mL.




Figure 7. Identifying limbal stem cells (LSCs) in a population of differentiated corneal cells. (A) Untreated cells have a side population of limbal stem cells (0.452% of total cells) with decreased Vybrant® DyeCycle™ Violet fluorescence due to ABCG2-mediated dye efflux. (B) The percentage of cells in the side population is greatly reduced (0.023% of total cells) when the ABCG2 membrane pump is inhibited with 5 μM fumitremorgin C, preventing efflux of Vybrant® DyeCycle™ Violet.


Cell labeling procedure

1. Place 1 mL of cells (at 1 x 106 cells/ mL in KSFM) in each of two 12 x 75 mm tubes, labeled “control” and “test”.
2. Add fumitremorgin C to the tube labeled “control” to a final concentration of 5 μM.
3. Add 1 μL Vybrant® DyeCycle™ Violet per mL of cells to create a 5 μM staining concentration.
4. Incubate 90 min at 37°C, protected from light.
5. Wash cells: add 3 mL KSFM to tubes, centrifuge at 100 x g for 5 min, discard supernatant, and resuspend in 1 mL KSFM for analysis.

Analysis on the Attune® Acoustic Focusing Cytometer

1. Verify instrument performance with Attune® Performance Tracking Beads.
2. Create a new experiment.
3. Open the workspace by double-clicking on the first sample in the experiment browser.
4. Create two dual-parameter plots for VL1-A vs. VL1-H and VL1-A vs. VL3-A.
5. Use the “test” sample to optimize PMT voltage settings so that events can be seen on all histograms. Collect 10,000 events from this sample.
6. Create a polygon gate around the singlets population in the VL1-A vs. VL1-H density plot.
7. Select your first sample tube of stained cells.
8. Set the acquisition volume, collection rate, threshold, and recording criteria as desired. For this example, we used a 1,000 μL acquisition volume, 200 μL/min collection rate in Standard Mode, default FSC threshold at 10,000, and used the “record continuously” setting. Acquisition of at least 100,000 events is recommended.
9. Proceed with recording data for samples as shown in Figure 7.


Conclusion

Vybrant® DyeCycle™ Violet side population analysis can be used to identify and characterize stem cells as a single-color assay or in combination with a multicolor panel of antibodies on the Attune® Acoustic Focusing Cytometer. The high-throughput capabilities of the Attune® Acoustic Focusing Cytometer provide a powerful method to identify rare populations such as hematopoietic and tissue-specific stem cells.

Back to top

References

1. Goodell MA, Brose K, Paradis G et al. (1996) Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 183:1797–1806.
2. Goodell MA, Rosenzweig M, Kim H et al. (1997) Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat Med 3:1337–1345.
3. Telford WG, Frolova EG (2004) Discrimination of the Hoechst side population in mouse bone marrow with violet and near-ultraviolet laser diodes. Cytometry A 57:45–52.
4. Ema H, Morita Y, Nakauchi H et al. (2005) UNIT 22B.1: Isolation of Murine Hematopoietic Stem Cells and Progenitor Cells. Curr Protoc Immunol 22B.1.1–22B.1.13.
5. Telford WG, Bradford J, Godfrey W et al. (2007) Side population analysis using a violet excited cell-permeable DNA binding dye. Stem Cells 25:1029–1036.
6. Telford WG (2010) UNIT 9.30.1, Supplement 51: Stem Cell Side Population Analysis and Sorting Using DyeCycle Violet. Curr Protoc Cytom 9.30.1–9.30.9.
7. Baumgarth N, Roederer M (2000) A practical approach to multicolor flow cytometry for immunophenotyping. J Immunol Methods 243:77–97.
8. Maecker HT, Trotter J (2006) Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A 69:1037–1042.

Back to top

For more than a decade, Life Technologies has provided you with key resources to address challenges in stem cell research. Now we’ve harnessed the power of these innovations to offer a total system covering the entire research spectrum.

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. © 2012 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.