Introduction
Autophagy is the mechanism of the cell that disassembles unnecessary proteins and organelles to recycle or metabolize them. When placed in certain physiological conditions, the cells produce phagophore to cover unnecessary components and form autophagosome. The autophagosome fuses with lysosome (production of autolysosome), and acid hydrolase from lysosome degrades the unnecessary components. Autophagy has been confirmed to be related to cancer and neurodegenerative diseases such as Alzheimer and Parkinson’s disease, and as possessing a physiological function in aging.
The following are the results of experiments using the CQ1 for imaging and the high content analysis software CellPathfinder for analysis. In this experiment, DALGreen-Autophagy Detection (Donjindo Molecular Technologies, Inc.)*1 was used; it penetrates cell membranes and is drawn into autophagosome along with unnecessary components, fusing with lysosome and then increasing fluorescence in the acid environment in autolysosome.
Experiment Procedure
- HeLa cells were cultured in 96-well plates (Greiner #655087) for 24 hours.
- DALGreen – Autophagy Detection was added according to the attached protocol and incubated for 30 minutes.
- The cells were washed in the cultured medium, and then medium were changed to normal medium, autophagy inducer medium (not containing amino acid) and autophagy inhibition medium (Bafilomycin added to the inducer medium at the final concentration of 100nM). Time-lapse imaging using the CQ1 was implemented every 30 min, for 6 hours (object lens magnification of 20x, 4 fields, 6 Z-slices per well), and fluorescence images processed with DALGreen (Ex:405nm/Em:500-550nm) and bright field images were captured.
- Contrast-enhanced Bright Field (CE Bright Field) images*2 were created from the bright field images using the CellPathfinder, and the cells were counted. Granules produced through autophagy were analyzed in the fluorescence images.
Fig. 1: Autophagy detection Fluorescence images of the control (A), autophagy induction (B) and autophagy inhibition (C) 6 hours after changing the medium.
(D) (E) (F): CE Bright Field images of (A), (B) and (C) respectively (Scale bar: 100 µm)
(G): Images (from left) immediately after changing the medium, 3 hours later and 6 hours later (Scale bar: 50 µm).
Time lapse movie : Play
Fig. 2: Autophagy analysis result Merged images of the control (A), autophagy induction (B) and autophagy inhibition (C) 6 hours after changing the medium.
(D) (E) (F): Analyzed images of (A), (B) and (C) respectively (Blue points: centers of recognized cells; Red: recognized regions of autophagy granules) .
(G) (H): Change of count and total area of granules per cell over time. Error bars indicate SE (n=3). (Blue: control, Red: autophagy induction, Green: autophagy inhibition).
The count and total area of granules increased over time only in the autophagy induction.
Results and Discussion
It was confirmed that live cell autophagy can be easily observed using the CQ1 and DALGreen-Autophagy Detection. Also, autophagy was induced in the media not containing amino acid and inhibited by adding Bafilomycin. The CQ1 allows for the observation of changes with time while maintaining the culture environment through the use of its stage heater for the control of temperature and humidity, as well as the concentrations of CO2 and O2 through the combined use of a gas mixer.
*1 Reference: Iwashita et al., Small fluorescent molecules for monitoring autophagic flux, FEBS Lett,2018,592(4),559.
*2 CE Bright Field images
Contrast-enhanced Bright Field (CE Bright Field) images refer to processed images emphasizing the cell thick areas (Fluor type) and contours and details (Phase type), which are suitable for cell recognition without staining.
Our Social Medias
We post our information to the following SNSs. Please follow us.
Follow us | Share our application | |
@Yokogawa_LS | Share on Twitter | |
Yokogawa Life Science | Share on Facebook | |
Yokogawa Life Science | Share on LinkedIn |
Yokogawa's Official Social Media Account List
相關產品&解決方案
-
CellPathfinder高內涵影像分析系統專用軟體
CellPathfinder 是專為Yokogawa HCA 系統、CQ1 和 CellVoyager 系列而設計的軟體。 從初學者到專家,都可讓您量化細微的生理變化,使用各種圖形選項量化無標籤樣本。
-
CellVoyager高內涵分析系統CQ3000
CQ3000可以根據應用組合選項,在培養細胞的同時快速獲取高分辨率的3D圖像。
-
CQ1台式高內涵分析系統
CellVoyager CQ1採用節省空間的台式設計,提供高質量的共聚焦圖像和延長的活細胞成像。
-
CV8000 High-Throughput System 高內涵影像分析系統
CellVoyager CV8000 是一款高階的高內涵分析系統,使用橫河特有的高速共軛焦掃描儀。具備水鏡、多達四個高視野相機、細胞培養環境的載物台和自動化分液器的組合,不僅實現高內涵、高解析,也可以使用更複雜的評估系統進行表型篩選。
-
High Content Analysis CellVoyager
我們的高內涵分析 (HCA) 系統使用功能強大的軟體,支援從基礎科學到復雜化合物篩選的廣泛研究應用。
-
Life Science
Yokogawa 的螢光顯微影像系統和生命科學解決方案支援從基礎研究、研發藥物到臨床前試驗的應用。
Yokogawa的高內涵影像篩選系統和雙轉盤式共軛焦技術應用於再生醫學、研發藥物和精密醫學,實現高速、高辨識度的活細胞成像。