staining mouse bone marrow cells with hoechst to detect side population cells by flow cytometry

サイドポピュレーション細胞の検出を容易にする代替レーザー励起法

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 &#160;CD117+Sca-1+Lin-Thy1low&#160;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/&#946;-catenin, TGF-&#946;, 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&#160;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.

著者スポットライト:サイド集団細胞を分析するためのヘキスト染色のための革新的なレーザー技術

フローサイトメトリーによるHoechst側集団細胞の効果的な検出

マウス骨髄細胞をHoechstで染色し、フローサイトメトリーによるサイドポピュレーション細胞の検出

staining-mouse-bone-marrow-cells-with-hoechst-to-detect-side

Research

JoVE Journal

Cancer Research

Staining Mouse Bone Marrow Cells with Hoechst to Detect Side Population Cells by Flow Cytometry

182 ビュー. まず、マウスから骨髄細胞を採取し、2ミリリットルの細胞懸濁液に1ミリリットルあたり5マイクログラムのHoechst 33342を加えます。ネガティブコントロールチューブも用意します。30分ごとに穏やかに攪拌しながら摂氏37度の水浴で90分間インキュベートしますインキュベーション後、細胞を氷上で5分間冷却し、次に摂氏4度で250Gで5分間遠心分離?...

ビデオ: マウス骨髄細胞をHoechstで染色し、フローサイトメトリーによるサイドポピュレーション細胞の検出

Effective Detection of Hoechst Side Population Cells by Flow Cytometry

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.

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 ...