CQ1 Applications

Applications: CQ1

• Cell cycle
• Spheroid
• CTC
• Granule
• Scratch assay
• Cell Proliferation
• Induction of differentiation
• Time lapse

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Cell cycle

Cell cycle analysis: M-phase inhibitor

Cell cycle analysis in relation to H3Ser10P immunofluorescence by utilizing the CQ1ʼ s multi-color channel capabilities. Histone molecules are phosphorylated during cell cycle progression with phosphorylation of the 10th serine of histone H3 being one of the well-characterized events of late-G2 to M progression.

Cell cycle

3D imaging of confluent A549 cell(lung carcinoma)

Protocol

Image data		Recognition		Total intensity Draq7 in DNA

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Spheroid

Spheroid analysis

The CQ1 conducted the 3D imaging and 3D analysis for the nuclei of canceration stem cells dyed with Draq5. The measurement data from the number of cells and size can lead to the analysis of differentiation status.

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CTC

CTC（Circulating tumor cells）

CTCs are tumor cells that circulate in peripheral blood. Developed tumors metastasize through the bloodstream and lymph fluid. Therefore, tumor cells exist in the bloodstream when metastasis occurs. The detection of CTCs makes it possible to diagnose recurrence and metastasis at an early tumor stage. CTCsʼ numbers are very small as only less than 100 CTCs are contained in more than 1x106 of blood cells in 10 ml of cancer patientʼs blood. Therefore it is difficult to detect CTCs with a flow cytometer because they detect CTCs as noise. However, it is very easy to detect rare CTCs with an Image cytometer.

A. Marker for epithelial cells (AE1/AE3: FITC label)
B. Marker for tumor stem-like cells（CD133: Texas Red label）
C. Marker for both epithelial cells and tumor stem-like cells
(AE1/AE3*CD133)

Data:  Hisae Iinuma, ph.D, Department of Surgery, Teikyo University school of medicine

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Granule

Granule analysis: gamma-H2AX focus formation

The phosphorylation of histone H2AX Ser139 (gamma-H2AX) is one of the significant events upon DNA double-strand break. Quantitative measurement of gamma-H2AX focus formation can be easily performed by using the high-speed confocal image acquisition in combination with the Granule Analysis Template.

Granule analysis (GPCR)

GPCR(G-Protein Coupled Receptor）that exists on the cell membrane plays the role to transmit the signal from the outer cell to inter-cell through the G-Protein. Combining with GPCR, Ligand is absorbed from the membrane to inter-cell and forms the endosome, and desensitizes with neurokinin-1 receptor (NK1R). Internalization: By counting the number of intracellular vesicles (granule), you can carry out the drug screening and the function analysis.

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Scratch assay

Scratch assay
The scratch assay is one of the most commonly used methods to evaluate the cellular capability of infiltration and proliferation. The multicolor time-lapse imaging by CQ1 offers the quantitative analysis of cell migration and proliferation with time.

The scratch was created on the cultures of Fucci-HeLa cells. Mitomycin C (MMC) was added to the culture medium to interrupt the cell division cycle then washed out. Time-lapse imaging was performed for three days.
The scratch was infiltrated by the cells in G1 and S/G2/M phase in the control cultures, indicating that the scratch was infiltrated not only by cell migration but also by proliferation as well. On the other hand, most of the infiltrating cells were in the S/G2/M phase in the MMC-treated culture, indicating that the scratch was filled mostly by cell migration.

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Cell Proliferation

Cell Proliferation
The whole view of one well of a 96 wallplate can be captured as a single image by using a 2x objective lens. It is possible to rapidly judge cell growth in each well based on cell numbers or individual cell areas.

Image data

Recognition

 
Area                                                                          Mean intensity
 

Whole well in one shot by 2x lens

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Induction of differentiation

Quality control: human iPSC sphere
Sphere shape and pluripotency marker expression level are suitable indices for evaluation of the quality of pluripotent state of human iPSC sphere.
Data:  ReproCell

Whole well tile image (x2 lens)

Uniform pluripotent state                   Non-uniform sphere

 
X Axis:  DNA amount per sphere                  X Axis:  Sphere size (area)
Y Axis:  Oct3/4 expression                            Y Axis:  Oct3/4 expression

Quality control: Induction of differentiation
Aggregated cells images were taken a 3D manner. By image analysis marker expression level as well as spatial information of individual cells were quantified.

Marker expression per cell

 
Differentiation marker positive               Differentiation marker negative

   
Undifferentiated        Partially differentiated   Partially differentiated

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Time-lapse

Time-lapse analysis: ES colony
Time-lapse analysis of colony size and individual cells allows monitoring of colony formation states. CQ1ʼs image can perform image acquisition with low photo-toxicity.
Data:  Kyoji Horie, PH.D, Physiology II, Nara Medical University

ES colony, excerpt from time-lapse images taken at 30 min. interval.

Time-lapse analysis: Apoptosis

Spread HeLa cells to 96well microplate with 10,000 cells/well. Stain with Hoechst33342 (1µg/ml, 30 min, 37 ℃) and treat with Staurosporine (0 - 10µM) and capture image every 15 min. Recognize DNA fragmentation area of nuclei at Staurosporine 10µM treatment.

Time-lapse movie
play

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