Introduction
PKC(protein kinase C) is family of protein kinase enzymes which locates downstream of receptor Tyrosine kinase and G-protein coupled receptor, .involved in phosophorylation of various substrates. PKC is involved in wide range of physiological functions, such as cell proliferation, gene expression, receptor regulation, ion channel gating, apoptosis, and the other physiological functions via these substrates and play a central role in intracellular signal transduction. It is known that PKC is activated by phorbol ester typified by PMA (phorbol 12-myristate 13-acetate) and migrates from the cytoplasm to the cell membrane. Here, we introduce the result of imaging and analysis of PKC translocation using PMA treated HeLa cell by using CellVoyager CV8000 and Yokogawa analysis software.
Results
All images were captured by using Yokogawa CV7000 and PKC location changes were analyzed by “Plasma Membrane Translocation” method of analysis software (Fig.1). As a result, PKC particles increased on the cell membrane depending on the PMA concentration, and a PMA dose response curve could be generated from the change in the fluorescent intensity of PKC in the plasma membrane and cytoplasm. (fig2a, 2b). Also, with Yokogawa analysis software, individual particle area and intensity were also generated, so we created a graph based on these data using Spotfire®(Fig.3, Fig.4). In this way, using “Plasma Membrane Translocation” algorithm, we can analyze and describe various protein translocation of nuclear, cytoplasm, and plasma membrane in detail.
Fig.1 Original images acquired using CV7000, and recognized image.
(Blue: Nuclear Hoechst33342, Green: PKC Alexa Fluor488)
PMA(1000ng/ml) treated wells show PKC translocation to plasma membrane.
In the recognized image, 3μm inner from cytoplasm outline is recognized as plasma membrane area.
Experimental
- HeLa cells were seeded at 10,000 cells/well at 96 well plate.
- PMA treatment for 20min. (final conc : 0～1000ng/ml)
- Fixed by formaldehyde.
- Immunostain PKC, and stain nuclei with Hoechst33342.
- Acquire image with CV7000.
・Objective lens : 10X
・4 images/ well
・Exposure time : Hoechst33342 (405nm) 200 msec Alexa Fluor488 (488nm) 400 msec
Analysis Method
“Plasma Membrane Translocation(Basic Mode)” is used for image analysis. Nucleus recognized by Hoechst33342 staining, cytoplasm and the PKC particle are recognized by Alexa Fluor488 derived fluorescent. Analysis algorithm parameter was modified to detect certain intensity range and area of the object to remove artifact. Plasma membrane was defined as 3um inner from outline of cytoplasm region.
Fig. 2 PMA dose response curve
(a) Total intensity of particle present in the plasm membrane per cell
(b) Intensity ratio (Plasma membrane/Cytosol) of the plasma membrane and cytoplasm
Fig. 3 Total intensity ratio histogram of particle present in the plasma membrane and cytoplasm.
(Plasma membrane/Cytoplasm)
Fig. 4 Total intensity ratio of particle at plasma membrane and cytoplasm at each PMA concentration.
(Plasma membrane/Cytosol)Blue color is ≧0.97cell.
Spotfire® is a registered trademark of TIBCO Software Inc.
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
相关产品&解决方案
-
CellPathfinder
CellPathfinder is designed for our HCA systems, CQ1 and the CellVoyager series. From beginners to experts, the analysis software lets you quantify subtle physiological changes and even label-free samples with various graph options.
-
CellVoyager高内涵分析系统CQ3000
CQ3000可以根据应用组合选项，在培养细胞的同时快速获取高分辨率的3D图像。
-
CQ1台式高内涵分析系统
CellVoyager CQ1采用节省空间的台式设计，提供高质量的共聚焦图像和延长的活细胞成像。
-
CV8000高内涵筛选系统
CellVoyager CV8000是先进的高内涵筛选系统。改进的内置培养箱可以让客户分析长期的活细胞反应。凭借其可扩展性、4个摄像头、5个激光器和可选的内置移液器，该系统允许日益复杂的分析开发和高内涵筛选。
-
生命科学
解锁生命本质
横河电机灵敏、高精度的解决方案可实现快速准确的测量，支持突破性的研究，加快药物发现，并优化大规模生物生产。
-
高内涵分析
我们的高内涵分析系统利用强大的分析软件助力从基础科学到药物发现筛选的一系列研究应用。
