Effective Detection of Hoechst Side Population Cells by Flow Cytometry

Podsumowanie

Here, we show that high-power 375 nm and 405 nm lasers can effectively excite Hoechst 33342 and serve as a viable alternative to the 355 nm laser for side population (SP) cell detection, thereby expanding the range of available lasers in flow cytometry applications.

Streszczenie

The side population (SP) cells are identified through Hoechst 33342 staining and analyzed using flow cytometry (FCM). The Hoechst SP method is utilized for the isolation of stem cells based on the dye efflux properties of ATP-binding cassette (ABC) transporters. The method was initially employed for the identification and isolation of hematopoietic stem cells (HSCs), but it has now evolved to primarily focus on the identification and isolation of cancer stem cells (CSCs). The traditional detection method of FCM uses a 355 nm laser to excite the dye to detect SP cells. Through this study, we have successfully identified alternative approaches for dye excitation that can effectively replace the detection of SP cells using a 355 nm laser. This is achieved through the utilization of high-power 375 nm or 405 nm lasers. This allows us to exercise enhanced selectivity in the detection of SP cells rather than being solely limited to the 355 nm laser flow cytometry.

Wprowadzenie

The side population (SP) cells are identified through Hoechst 33342 staining and analyzed using flow cytometry (FCM). The SP cells are characterized by the pumping of the fluorescent DNA dye out of the cells through their ATP-binding cassette (ABC) transporter^1,2. The method was originally established for isolating murine bone marrow hematopoietic stem cells (HSCs)¹. The bone marrow SP cells were enriched with a population of HSCs characterized by  CD117⁺Sca-1⁺Lin^-Thy1^low expression^3,4. Thereafter, the method was widely used to isolate and enrich stem cells from other tissues, including cardiac muscle⁵, liver⁶, lung⁷, kidney⁸, and forebrain⁹. In particular, the method has been applied to isolate cancer stem cells (CSCs) in the past decade. CSCs represent a small population of cells that possess the properties of tumor initiation, self-renewal, resistance to chemotherapy, and metastatic potential¹⁰. CSCs were initially identified in hematopoietic malignancies¹¹ and subsequently observed in various solid tumors such as the prostate¹², ovarian¹³, gastric¹⁴, breast¹⁵, and lung¹⁶ carcinomas. Despite the availability of various techniques for CSC identification, the SP technique remains a favored choice owing to its broad applicability across diverse tissues and cell lines. Moreover, it is a valuable technique that isolates CSCs using fluorescence-activated cell sorting (FACS)^16,17,18. The experimental findings demonstrate that SP cells exhibit pronounced tumorigenicity and display elevated expression levels of stem cell-associated genes^19,20. Our previous study²¹ has also demonstrated that transcriptomic analysis of isolated SP cells in multiple myeloma reveals enrichment of signaling pathways associated with stem cells, such as the hedgehog pathway. Meanwhile, we performed pathway enrichment analyses on the genes differentially expressed in the SP cells of acute myelogenous leukemia²². The altered genes were enriched in stem cell-related pathways (Wnt/β-catenin, TGF-β, Hedgehog, Notch). We found that 1 x 10⁵ SP cells could form tumors in BALB/c null mice²², whereas non-SP cells could not, indicating that SP cells had characteristics of leukemia stem cells. The efficacy of the Hoechst SP method in the identification of CSCs is evident.

The Hoechst SP protocol has been refined and enhanced through the progression of research. The protocol entails stringent control of the dye concentration, cell density, incubation temperature and duration, buffer composition, and pH value. The cell samples prepared in accordance with this protocol were subjected to flow cytometric analysis. Due to the excitation of Hoechst dye with a UV laser at 355 nm and detection of its fluorescence emission using both a 690/50 nm filter (Hoechst Red) and 450/50 nm filter (Hoechst Blue), a 350 nm laser is required for FCM. However, 350 nm lasers are not commonly equipped in most FCMs because of their high cost. Hence, we attempted to find an alternate approach for dye excitation for the effective detection of SP cells by flow cytometry. In this study, the capability of the 375 nm and 405 nm lasers in detecting SP cells was assessed and compared with that of the 355 nm laser. Our findings demonstrate a remarkable similarity between the SP cells detected by the 355 nm, 375 nm, and 405 nm lasers. These results suggest that the high-power 375 nm and 405 nm lasers can serve as feasible alternatives to the 355 nm laser for SP cell detection. The inclusion of additional excitation light sources for Hoechst 33342 facilitates the use of more flow cytometry models.

Protokół

The experiments in this study utilized a total of 10 C57BL/6 mice aged between 8 and 12 weeks. Experimental operations were conducted in accordance with a protocol that was approved by the Institutional Animal Care and Use Committee of Sichuan University (#201609309). Experimental materials and the parameters of flow cytometry used in this article are listed in the Table of Materials.

1. Isolation and collection of mouse bone marrow cells

1. Euthanize mice in strict accordance with the institutional guidelines. To induce unconsciousness in the mouse, administer inhalation anesthetics starting with 2% isoflurane, followed by a gradual increase in dosage to 5% isoflurane until cessation of respiration.
2. Dissect the mice in the surgical suite or the Fume cupboard. Clean and sterilize the mice with 70% ethanol.
3. Cut the skin and muscle of the leg with sharp scissors completely and dissect the femurs.
4. Crush the femur gently with a grinding rod in a mortar and flush out bone marrow cells with DMEM medium until the bone turns white.
5. Filter the obtained bone marrow cells with a 100 µm filter into 15 mL tubes. Centrifuge at 800 x g for 5 min at 4 °C.
6. Discard the supernatant and lyse residual red blood cells with 1 mL of red blood cell lysis buffer for 1 min at 4 °C. Centrifuge at 800 x g for 5 min at 4 °C. After centrifugation, wash again with DMEM medium.
7. Resuspend the cells in 2 mL of incubation solution (DMEM medium + 5% FBS), count the cells with an automatic cell counter and adjust the cell concentration to 1.0 x 10⁶ cells/mL with incubation solution.

2. Cellular staining

1. Supplement 2 mL of bone marrow cell suspension from each mouse with 5 µg/mL Hoechst 33342. To another 1 mL aliquot, add 100 µM verapamil as a negative control.
2. Incubate in a water bath at 37 °C for 90 min with gentle agitation every 30 min.
3. After incubation, cool the cells on ice for 5 min and then centrifuge at 250 x g for 5 min at 4 °C. Resuspend the cells in a cold running solution (HBSS + 2% FBS). Centrifuge at 4 °C for 5 min and discard the supernatant.
4. Resuspend the resulting cell pellet in 500 µL of running solution per sample. Prior to FCM analysis, supplement cells with 2 µg/mL of propidium iodide (PI) and place on ice for approximately 5 min.

3. Flow cytometry

NOTE: For a summary of the various systems and configurations used, please see Table 1.

1. Calibrate the Coefficient of variation (CV) values of the required channels using daily quality control (QC) fluorospheres in the FCMs and perform sample testing following the successful completion of quality control.
   1. Select the desktop shortcut of software and launch the software.
   2. Select Start QC/Standardization in the QC/Standardization menu to access the QC experiment.
   3. Insert the QC fluorospheres sample tube into the tube holder.
   4. Select Start to load the sample and begin to run the QC procedure. FCMs are ready for use after QC passes.
2. Excite the Hoechst 33342 dye of the same samples with a 355 nm, 375 nm, and 405 nm laser, respectively, for detection of Hoechst red in the 690/50 nm channel and Hoechst blue in the 450/50 nm channel.
   1. Create a new experiment by selecting New Experiment in the File menu, specify the file path, and save the experiment.
   2. Select Set Channel in the Settings menu. Select the channel signal check box (Y585, V450, V660, NUV450, NUV660, UV450, UV660), then add the reagent name in the Label column (Y585: PI; V450: Hoechst Blue-405 nm; V660: Hoechst red-405 nm; NUV450: Hoechst Blue-375 nm; NUV660: Hoechst Blue-375 nm; UV450: Hoechst Blue-355nm; UV660: Hoechst Blue-355 nm).
   3. Click Pseudo Color Plots icons in the plot area to create plots. Select an axis name to change which channel is displayed.
   4. Click Add Tube in the Test Tube screen to create new sample tubes and change their names.
   5. Select Run to load the sample, view the plots, and establish the gates. Adjust the gain and threshold settings. Select Record to save the data.
3. Design the gate setting logic.
   1. For the first plot, click the X-axis to select FSC-W and click the Y-axis to select FSC-A. Select Polygon Gate to draw gate A to circle the individual cell and exclude adherent cells (Figure 1A).
   2. For the second plot, click the X-axis to select FSC-A and click the Y-axis to select SSC-A. Select Polygon Gate to draw gate B to separate non-fragmentary cells and exclude cellular debris (Figure 1B).
   3. For the third plot, click the X-axis to select PI-A and click the Y-axis to select SSC-A. Select the Polygon Gate to draw gate D. Select Live cells exhibiting negative PI (Figure 1C) and draw gate C to obtain live cells.
   4. For the fourth two-dimensional plot, click the X-axis to select Hoechst Red and click the Y-axis to select Hoechst Blue. Right-click the Plot and select Property from the drop-down menu. Select Linear Format for both the X-axis and Y-axis. Select Polygon Gate to draw gate SP to get SP cells (Figure 1D).
4. To assess sample eligibility, use 355 nm of a specific flow cytometer²³ for SP cell detection.
5. Use the 355 nm (laser power: 20 mW) and 405 nm (laser power: 80 mW) lasers simultaneously to detect both the control cells (with added verapamil) and experimental cells.
6. Acquire fluorescence signals at the 690/50 nm and 450/50 nm channels corresponding to the two lasers. Observe the effective stimulation of Hoechst 33342 dye by 355 nm and 405 nm lasers with clear SP cells (Figure 2B-C).
7. For the same samples, use the 375 nm (laser power: 60 mW) and 405 nm (laser power: 80 mW) lasers for cell detection.

Wyniki

In Figure 2, the control group was treated with verapamil, which blocks ABC transporters in stem cells to prevent the elimination of Hoechst 33342. Thereby, the stem cells in the non-verapamil group expel Hoechst 33342 and form a negative cell population known as SP cells. The 355 nm laser effectively excited the Hoechst 33342 dye, resulting in clear observation of SP cells of bone marrow (Figure 2A). The SP cells of the same samples detected by the 355 nm laser...

Dyskusje

We used the protocol described to conduct three experiments, with each trial involving 3-4 mice, resulting in a total of 10 mice. The proportion of SP cells ranged from 0.05% to 0.76%. It is important to note that individual variations were observed among the mice. We utilized four flow cytometers to analyze the Hoechst samples. It is observed that the excitation of Hoechst dye by a 20 mW 355 nm laser on the new version of flow cytometry is equivalent to that of a 100 mW 355 nm laser on old-fashioned flow cytometry. This...

Materiały

Name	Company	Catalog Number	Comments
Automatic cell counter	Countstar	1M1200
Cell Filter(100 µm)	BIOFIL	CSS-013-100
Daily quality control fluorospheres	Beckman Coulter	B5230
Dulbecco's Modification of Eagle's Medium with 4.5g/L glucose (DMEM medium)	CORNING	10-013-CVRC
Fetal bovine serum	CORNING	35-081-CV
HBSS	Hyclone	SH30030.02
Hoechst 33342	Sigma-Aldrich	B2261
Propidium iodide (PI)	Sigma-Aldrich	P4170
Red blood cell lysis buffer	Beyotime	C3702
Verapamil	Sigma-Aldrich	V4629