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Investigating cytotoxicity by using Countstar FL image cytometer

Cytotoxicity assays are routinely used in many laboratories for a variety purposes from assessing the health of cell cultures to evaluating the toxicity of a panel of compounds. The measurement tool employed for these assays needs to be reliable, easy to use and relatively fast. The Countstar FL system (Fig 1) is a smart, intuitive cell analysis instrument that streamlines a wide variety of cellular assay including transfection, apoptosis, cell surface marker, cell viability and cell cycle assessments. The system provides robust fluorescence quantitative results. The easy-to-use, automated procedure guide you to complete a cellular assay form 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 Apoptosis kit

 

Other materials:CHO cell line (ATCC)、Puromycin (Gene operation)

 

Methods:

Cell growth medium

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

 

Drug treatment conditions

1.    CHO cells were plate in 6-well plates at 200,000 cells/well in medium containing DMEM, 2mM L-Glutamine, 1.5g/L sodium Bicarbonate and 10% FBS 24 hr.

2.    Add the drug for different concentration and incubated for 48 hr in 37℃ 5% CO2 humidified incubator.

3.    Harvest the cells.

 

Staining Procedure:

1. Prepare 0.3-0.5 million cells for next step;

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

3. Mix the samples gently and spin down cell sample at 400g for 3-5 minutes, aspirate medium, and then resuspend cells in 100µl binding buffer;

4. Add 1.5µl of Annexin V-FITC;

5. Add 4µl of 7-AAD solution, gently pipette the cells up and down ten times;

6. Incubate for 10-15min at RT (25℃) in the dark;

7. Spin down cell sample at 400g for 3-5 minutes, aspirate medium, and the resuspend cells in 100µl binding buffer; analyze by Countstar FL

 

Imaging and analysis with Countstar FL

1.    A dual-color application procedure was created by setting Green and Red channels to image FITC, 7-AAD fluorescence.

2.    3 fields were captured from each chamber, every sample was test for two times.

3.    After imaging and initial analysis were complete, data were exported and analyzed by FCS software.

 

Determining the does response curve for puromycin selection of CHO cells

Typical antibiotic working concentration range for mammalian cells is 0.5-10 µg/ml for puromycin. A range of increasing amounts of puromycin (0µg/ml -45µg/ml) were used across the plate as indicated (Fig. 2).


Figure 2. Signal well containing puromycin (0, 0.5, 1, 2, 3, 4, 5, 15, 45 µg/ml) are indicated.

 

Imaging and analysis with Countstar FL imaging system

A dual-color application procedure was created by setting Green and Red channels to image FITC, 7-AAD fluorescence, plus a bright field. Bright field picture reference segmentation was applied as a mask to sample the FITC fluorescence signal and 7-AAD. Example images of 0µg/ml and 45µg/ml are shown in Figure 3.


Figure 3. Images of 0µg/ml and 45µg/ml treated CHO cells. Left. 0µg/ml sample, show little fluorescence signal; representative image shown. Right. 45µg/ml sample, most of the cells stained with both FITC and 7-AAD.



Kill curve for puromycin selection of CHO cells

The results were analyzed by FCS5 image software, gating the cells dependent on the cell population and fluorescence intensity (Fig. 4A), the FCS express also supplies the function to review every signal cell, validation the data through the image (Fig. 4B). Graphpad prism5 was implemented to evaluate the IC50 of puromycin.



Figure 4. Kill curve assay showing identification of cell populations and results displayed as a scatter plot A. Images in dual channels were visually assessed to set a gating threshold to identify live, apoptotic, necrosis cell populations, and results were analyzed by FCS5image software. B. Review every signal cell in different phase and validate the data through the image. C. Results indicating a more necrosis rate at increased levels of puromycin.

 

Summary:

The Countstar FL system provides a rapid and easy means of evaluating cytotoxicity. FCS express supplies the function to review every signal cell, validation the data through the image. In addition, it combines multiple functionalities and is a compact, application-driven, automated cell imaging system providing robust quantitative results through the use of 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. In addition, Countstar FL can provide quick answers to a cytotoxicity question prior to scaling up to a more extensive high throughput screening experiment.