Analysis of intracellular Calcium Response Depending on Ionomycin Concentration

下載 (432 KB)

Introduction

Changes in intracellular Ca2+ concentration are an important analysis target in the study of intracellular signal transduction related to physiological phenomenon, such as muscle cell contraction. The following are the results of imaging and analysis of the increase of intracellular Ca2+ concentration in A10 cells, using the CellVoyager, by treatment of ionomycin, a Ca2+ ionophore.

Analysis Results

Using Ca2+ probe, Fluo4, images were captured using the CellVoyager (Fig. 1a~1c), and temporal changes in intracellular Ca2+ concentration were analyzed with its analysis software. The analysis software allows for the recognition of individual cells, thus enabling data acquisition for temporal changes in individual cells (Fig. 2a). Therefore, it is possible to exclude cells with abnormal reactions and analyze cells with pathognomonic reactions only. In addition, by varying the type and concentration of reagents for each well, variation in reactions can be analyzed (Fig. 2b, 2c).
)Images at 0.2 sec. interval extracted from the images of 2.6 sec. after start up to 5.4 sec. Ionomycin was added at 3.0 sec. from the start.(Red-framed image)
1(b)
Time lapse

Fig. 1a:Images taken at 0.2sec intervals, from 2.6sec to 5.4sec after the start of imaging. Ionomycin stimulation was started 3.0sec after the start of imaging (the image in red frame).
Fig. 1b: Time lapse movie

Average fluorescence intensities of individual cells in each well

Fig. 2a: Kinetic analysis of Fluo4 bright intensities of individual cells in each well

Average of the average fluorescence intensities for individual cells in each well

Fig. 2b: Kinetic analysis of average Fluo4 bright intensities in each well

Average of the average fluorescence intensities for individual cells at varying concentrations

Fig. 2c: Kinetic analysis of average Fluo4 bright intensities at various ionomycin concentrations

*For all graphs above, the fluorescence intensity at 2.9sec after the start of imaging is converted to zero as a reference.

Experiment procedure / Analysis method

Preparation of reagents

A10 cells were seeded on 96-well plates at the ratio of 10,000 cells/well and cultured for 24 hours, then a CO2 indicator, Fluo4, was added (Final concentration 1μM, reaction time: 20min), to monitor intercellular Ca2+-influx.

Imaging

Time lapse images were captured using the CellVoyager CV6000 under the following conditions.
Ionomycin was added to the cells using the built-in pipettor.

  • Imaging
    • Magnification: 20x objective lens
    • Exposure time: 100msec
    • Imaging interval: 100msec over a period of 20sec
    • Ionomycin dripping: 50μM 3sec after the start of imaging
    • Ionomycin final concentration: 0~5μM
    • Images captured per well: 1 Field(well center)
  • Ionomycin treatment conditions:
    • Air gap: 30μl
    • Aspirate volume: 50μl
    • Aspirate speed: 33μl/sec
    • Dispense volume: 80μl
    • Dispense speed: 22μl/sec

Ionomycin treatment conditions

Built-in pipettor setting (Kinetics Module)

Ionomycin was dispensed from each well of the source plate into each well of the assay plate (A10 cells) in one to one correspondence.
Dispensing setting

Image Analysis

The images were analyzed using the analysis software under the following conditions.

  • Graphs were created using Spotfire®.
  • Binarization: Sigma Threshold Object Diameter [25] Sigma [100]
  • Binary Transform: Opening Circle Circle Radius [7]
  • Labeling: Divide Each Region Watershed Threshold [50] Select Region For Area MinArea [640] MaxArea [999999]
  • Logical Operating: Region Operator Region1 [After Labeling] Operator Type [EXPAND] Region2 [Cytoplasm]

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
•Twitter @Yokogawa_LS Share on Twitter
•Facebook Yokogawa Life Science Share on Facebook
•LinkedIn Yokogawa Life Science Share on LinkedIn

 

Yokogawa's Official Social Media Account List

Social Media Account List


相關產品&解決方案

  • 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。

    更多
  • 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。

    更多

置頂