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Quantitative cell cycle analysis by using Countstar FL image cytometer

Measuring the incorporation of DNA-binding dyes has been a well-established method for determining cellular DNA content in cell cycle analysis. Propidium iodide (PI) is a nuclear staining dye that is frequently applied in measuring cell cycle. In cell division, cells containing increased amounts of DNA display proportionally increased fluorescence. Differences in fluorescence intensity are used to determine the DNA content in each phase of the cell cycle. The Countstar FL system (Fig. 1) is a smart, intuitive, multifunctional cell analysis instrument that can obtain precise data in cell cycle analysis and can detect cytotoxicity by cell viability assay. The easy-to-use, automated procedure guides you to complete a cellular assay from imaging and data acquisition.


Figure 1. Countstar FL system combines the functionalities of a digital microscope, an image cytometer and a cell counter in a single bench top instrument



Materials:

Countstar products:Countstar FL、Countstar Chamber slider、Countstar Cell cycle kit

Other materials:MCF7 cell line (ATCC)、Nocodazole (Sigma)

 

Methods:

Cell growth medium

MCF7 cells were cultured in complete growth medium (DMEM, 2mM L-Glutamine, 1.5g/L Sodium bicarbonate and 10% FBS).

 

Drug treatment

1.      MCF7 cells were seeded in 6-well plates at 300,000 cells/well in medium containing DMEM, 2mM L-Glutamine, 1.5g/L Sodium bicarbonate and 10% FBS. Incubate the plates for 24h at 37°C, 5% CO2 incubator.

2.      Replace old medium with seven different concentration of Nocodazole in fresh culture medium. The top concentration of Nocodazole was 400 nM, 2.5 folds serial dilution. Each sample has a duplicate well. Leave two wells as DMSO control and keep the final concentration of DMSO same as samples. Incubator the plates at 37°C, 5% CO2 for a further 16 h.

 

Staining Procedures

1.      Cell viability assay

Use trypan blue staining to detect the cytotoxicity of different concentration of Nocodazole. Old medium was removed, and the cells were washed once with PBS and digested with trypsin. Then 20ul cells of each sample were stained with 20ul trypan blue. Add 20ul mixture to Countstar chamber slide and count cell number by Countstar FL.

2.      Cell cycle analysis assay

Step 1. Prepare 0.3-0.5 million cells for cell cycle analysis according to the cell number in cell viability assay;

Step 2. Spin down cell sample at 400g for 3-5 minutes, aspirate medium, and then resuspend cells in 200ul PBS;

Step 3. Remove supernatant and fix cells with 200ul 60% ethanol at 4°C for 1 h or more;

Step 4. Centrifuge at 400 g for 3-5 min and remove supernatant. Wash once with 200ul PBS;

Step 5. Resuspend cells in 100ul cell cycle buffer containing 1ul PI solution and 0.5ul RNAse A. Incubate for 30min at 37℃ in the dark and detect samples with Countstar FL.

 

Imaging and analysis with Countstar FL

1.      Detect the cell number and cell viability by using “Trypan Blue Viability” directly.

2.      The cell cycle program was created by setting Red channel to image PI fluorescence with 3 fields captured from each chamber. After imaging and initial analysis completed, data was exported and analyzed by FCS software from De Novo. The process of cell cycle assay was described briefly in Figure 2.

 

Caution: PI is a potential carcinogen. It is recommended that the user wear protective clothing, gloves, and eye/face protection in order to avoid contact with skin and eyes.




Figure 2. Process of cell cycle assay



Assessing the cytotoxicity of Nocodazole in MCF7 cells

The cell viability assay is usually used in assessing anti-proliferative activity and cytotoxicity of compounds. The “Trypan Blue Viability” in Countstar FL was applied to assess the cytotoxicity of Nocodazole in MCF7 cells. From Figure 3, the cytotoxicity of Nacodazole was accurately measured by the percentage of Sample viable cells/Control viable cells. The IC50 of Nacodazole was evaluated by Graphpad prism5. 


Figure 3. The cytotoxicity of Nacodazole was measured by Countstar FL.

 


Quantitative cell cycle analysis with Countstar FL imaging system


Different DNA content in cell division can be predicted by determining the fluorescence intensity of PI which can intercalate with DNA. MCF7 cells were applied to analyze cell cycle arresting following treatment with different concentration of Nocodazole. The bright field is used to identify individual cells and PI channel is applied to accurately count signal cells within clumps (Fig. 4A). The results were analyzed by FCS Express 5 image software and the IC50 of Nacodazole was evaluated by Graphpad prism5. From Figure 4B, Nocodazole induced an obvious decrease in the G0/G1 phase and slightly reduction in S phase, while induced a sharp increase in the proportion of G2/M cells. The samples of DMSO control, 64nM and 400nM Nacodazole were chosen to present the intuitive results of cell cycle arresting in histogram (Fig. 4C).




Figure 4. The percent population of cells with or without Nacodazole treatment in different cell cycle phase were displayed in population histogram and dose response curve. A. Images were obtained from PI channel and Bright field from Countstar FL. B. Percent population of cells in different cell cycle phase varied with different concentration of Nacodazole. C. Population histogram for the DMSO control, 64nM and 400nM Nocodazole samples generated by FCS Express 5 software.

 

Summary:

The Countstar FL system is able to accurately analysis the dose response for cell cycle blockers by using a limited number of cells. FCS express software supplies similar function with FlowDraw to review percent population of cells in different cell cycle through the histogram. In addition, Countstar FL combines multiple functionalities and is a compact, application-driven, automated cell imaging system providing robust quantitative results by using preconfigured biological applications. Each pre-set assay is an easy-to-use, automated module that covers all steps of a specific biological assay to simplify routine cell laboratory tasks while providing high-quality scientific data. For quantifying cell cycle, Countstar FL supplies a sophisticated and reliable method.