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.
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.
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) transporter1,2. 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–Thy1low expression3,4. 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 genes19,20. 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.
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
2. Cellular staining
3. Flow cytometry
NOTE: For a summary of the various systems and configurations used, please see Table 1.
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 and the 405 nm laser were found to be essentially identical (Figure 2D).
From Figure 2 and Figure 3, we saw that other models of non-traditional flow meters can also identify SP cells. The results indicate that both 375 nm and 405 nm lasers effectively excited Hoechst 33342 to obtain SP cells (Figure 3A-B). When it is necessary to detect or isolate CSCs or HSCs, we can opt for a 375 nm or 405 nm laser instead of a 355nm laser.
Figure 1: Flow cytometry-based gate setting logic for detecting SP cells. (A) FSC-A and FSC-W were utilized to exclude adherent cells. Gate A represents the single-cell cluster. (B) The parameters of FSC-A and SSC-A were employed for the removal of cellular debris. Gate B represents the non-fragmentary cell cluster. (C) Live cells exhibiting negative propidium iodide (PI) were selected. Gate C represents the live cell cluster. (D) Fluorescence signals were acquired through the 690/50 nm (Hoechst-red) and 450/50 nm (Hoechst-blue) channels. Gate SP represents the SP cell cluster. Please click here to view a larger version of this figure.
Figure 2: High-power 405 nm laser detects SP cells as effectively as the 355 nm laser. (A) The Hoechst 33342 dye was excited by the 355 nm laser from a conventional flow cytometer to detect the SP cells of bone marrow. (B, C) The SP cells of bone marrow were identified by the 355 nm and 405 nm lasers of the same FCM. (D) The SP cells of the same samples detected by the 355 nm laser and the 405 nm laser were found to be essentially identical. Please click here to view a larger version of this figure.
Figure 3: High-power 375 nm and 405 nm lasers are capable of detecting SP cells. (A) High-power 375 nm laser can detect SP cells. (B) The SP cells of bone marrow can also be detected utilizing the 405 nm lasers. Please click here to view a larger version of this figure.
Laser type | Laser wavelength (nm) | Laser power (mW) | Hoechst red filter (nm) | Hoechst blue filter (nm) | |
Moflo Astrios EQ | Argon gas | 355 | 100 | 692/75 | 448/59 |
CytoFLEX LX | Semiconductor | 355 | 20 | 690/50 | 450/50 |
405 | 80 | 690/50 | 450/50 | ||
CytoFLEX S | Semiconductor | 375 | 60 | 690/50 | 450/50 |
CytoFLEX SRT | Semiconductor | 405 | 80 | 690/50 | 450/50 |
Table 1: Summary of flow cytometer configurations.
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 phenomenon can be attributed to the variation in laser types employed in both systems. The potential reason for the observed effect of 375 nm and 405 nm lasers could be attributed to the substantial power output of both lasers in conjunction with the utilization of the avalanche photodiode (APD) detector. The Hoechst Red-A channel captured the near-red long wavelength of 690 nm, where the photoelectric conversion efficiency of the photomultiplier tube (PMT) was relatively low. However, the APD demonstrated a photoelectric conversion efficiency approximately ten times higher than that of PMT24. Therefore, when combined with high-power 375 nm and 405 nm lasers, APD detectors efficiently distinguish SP cells. The 405 nm laser not only effectively detects SP cells but also exhibits superior capability in distinguishing a subset of weakly Hoechst Red positive populations compared to the 355 nm laser.
The critical step of the protocol is to use flow cytometry with a 375 nm laser or a 455 nm laser to detect cells with 690/50 nm (Hoechst-red) and 450/50 nm (Hoechst-blue) channels. While the technique detects SP cells with 375 nm and 405 nm lasers instead of 355 nm laser (traditional laser for analyzing SP cells1), which are more common in FCMs, high-power 375 nm and 405 nm lasers are required. This leads to the limitations of this technology. The significance of the method is to expand the range of lasers used to detect Hoechst dyes so that other models of flow cytometry can be used for SP cell detection.
The authors have nothing to disclose.
This work was supported by the grants to J.H. from National Natural Science Foundation of China (No. 81800207). The assistance of Beckman Coulter, Inc. in providing support for flow cytometry and parameter calibration is greatly appreciated. We thank Jiao Chen of the State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, and Yu Qi of Regenerative Medicine Research Center, West China Hospital, Sichuan University, for their assistance in flow cytometry data acquisition.
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 |