MitoSpy™ 系列粒腺體活細胞染劑 Mitochondrion Living Cell Dye 

MitoSpy™ 粒線體定位探針是可滲透細胞的螢光化學試劑，用於標記活細胞中的粒線體。
MitoSpy™ 根據其膜電位定位到粒線體，可用於指示細胞健康狀況以及定位，也可以被固定和透化以用於進一步的基於抗體的檢測。MitoSpy™ Orange CMTMRos 和 MitoSpy™ Red CMXRos 這兩種染劑適用於固定和透化的組織，亦可以與其他的抗體共染（co-stain）。

MitoSpy™ Orange 和 MitoSpy™ Red 含有一個氯甲基基團（chloromethyl group），以共價方式附著在細胞中的半胱胺酸（cysteines）上。這使它們在樣本固定（fixation）和透化（permeabilization）處理後更有可能保留在細胞中。然而，由於固定/透化過程會沖洗掉大量試劑，因此需要更高濃度的 MitoSpy™ Orange 和 MitoSpy™ Red 確保穩染色的穩定性。

在固定/透化後，大多數的 MitoSpy™ Green 和 MitoSpy™ NIR 將被沖洗掉。因此，在需固定/透化的樣本中不建議使用 MitoSpy™ Green 和 MitoSpy™ NIR 染劑。

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MitoSpy™ 細胞活染示意圖

HeLa cells were stained with 0.25µM CytoPhase™ Violet dye (red) for 60 minutes at 37°C. Then 20 nM of MitoSpy™ NIR DiIC1(5) (green) was added for an additional 30 minutes at 37°C. The Z-stack images were captured on an LSM 880 with Airyscan using a 63x Oil objective. The files were Airyscan processed and a Maximum Intensity projection was created using Zen software; image courtesy of the Biophotonics Core Facility at the Salk Institute.

NIH3T3 cells were stained with 100 nM of MitoSpy™ Red CMXRos (red) for 20 minutes at 37°C, fixed with 1% PFA for ten minutes at room temperature, and permeabilized with 1X True Nuclear™ Perm Buffer for ten minutes at room temperature. Then the cells were stained with Flash Phalloidin™ NIR 647 (green) for 20 minutes at room temperature and counterstained with DAPI (blue). The image was captured with a 60x objective.

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使用 MitoSpy™ 進行細胞活染

	Live staining  (50 nM)	PFA-fixed and permeabilized staining (500 nM)
MitoSpy™ Orange
MitoSpy™ Green
HeLa cells that were stained live with either MitoSpy™ Orange (yellow) or Green (green). Cells that were fixed and permeabilized with 4% PFA and 0.1% Triton X-100 were also stained with DAPI (blue). Photos were taken with a 60x magnification.

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MitoSpy™ 活染劑量建議

*請注意，所有MitoSpy™產品僅適用於活體樣本。以下建議反映了MitoSpy™是否能成功保留在「細胞處理」後。

BioLegend 昶安獨家代理

MitoSpy™ Flow Cytometry 染色示意圖

MitoSpy™ Orange、MitoSpy™ Red 和 MitoSpy™ NIR 也可用作細胞健康的指標。當細胞的粒線體正在進行主動的呼吸作用時，粒線體膜上會存在一個被稱為膜極化的電位差。如果細胞因為凋亡或細胞死亡而感到受損時，這個探針就不會強烈吸引到該細胞的粒線體。在下面的圖像中，對 MitoSpy™ Orange、MitoSpy™ Red 和 MitoSpy™ NIR 呈陽性的細胞是活躍且健康的，而對 Annexin V 呈陽性的事件處於凋亡的初期階段。在這種流式細胞儀應用中，MitoSpy™ Orange、MitoSpy™ Red 和 MitoSpy™ NIR 在分析前不應固定，因為固定會導致試劑的大量損失。低或負的 MitoSpy™ Orange、MitoSpy™ Red 或 MitoSpy™ NIR信號表明細胞具有凋亡表型。如果在固定過程中失去試劑，則信號強度的損失將混淆準確定性凋亡的能力。只有在成像應用中，MitoSpy™ Orange 和MitoSpy™ Red 才適用於亞細胞定位的固定。不建議使用 MitoSpy™ NIR 進行固定。

Human T-cell leukemia cell line, Jurkat, was treated for 5 hours with LEAF™ purified anti-CD95 (clone EOS9.1), then stained with an impermeant nucleic acid stain, APC, Alexa Fluor® 488, or Brilliant Violet 421™ Annexin V, and either MitoSpy™ Orange, Red, or NIR as indicated. Nucleic acid stain positive events were excluded from analysis.

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MitoSpy™ 產品資訊

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