Simple, Fast Determination of Viability by Staining Cells with Propidium Iodide

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Introduction

Cellometer Vision incorporates image based cell counting and fluorescence detection in a compact and easy-to-use instrument. Cellometer Vision is ideal for many cell population characterization assays such as rapidly determining viability by staining cells with propidium iodide.

Cell viability is routinely tested in cell culture laboratories and used in many assays such as cell proliferation, cytotoxicity, and tumor killing activity. In the traditional viability method of trypan blue exclusion, live cells exclude the dye as the membrane is impermeable, but dead cells uptake the dye due to the lack of an intact cell membrane. Viability is then determined by manually counting the stained sample in a hemacytometer, a time consuming and labor intensive process. More importantly, trypan blue stained samples have to be analyzed within 3-5 minutes, as longer incubation will increase blue colored cells. Trypan blue staining may result in an overestimate of viable cells especially for low viability samples.

Reliable and accurate viability testing can also be done using propidium iodide (PI), a fluorescent dye. Cellometer Vision was developed to automatically count total cells and propidium iodide fluorescence labeled dead/dying cells for fast, simple PI viability determination.

Using proprietary algorithms, Cellometer Vision's robust operating software accurately analyzes cell images, and generates this data in less than 60 seconds typically. Cell images and all analysis data, including cell size distribution histograms, can be saved or exported to Excel for further analysis.

Method

Direct PI labeling

1. Harvest cell from a 75 cm² flask and wash cell pellet with appreciated culture media.
2. Loosen cell pellet then re suspend cell sample with 10 ml of culture media.
3. Take 20µl of cell sample and mix cell with 20µl of PI solution.
4. Mix well by gently vortexing sample.
5. Load 20µl of sample into the disposable counting chamber.

Running Assay:

Results

Total counted Jurkat cells are indicated by green circles in the brightfield image (Figure 1). Dead cells stained with PI are indicated as fluorescent positive, green circled cells in the fluorescence image (Figure 2). Total and fluorescence positive cells counted, concentration, mean size, and percentage viability are displayed in the results box (Figure 3).

Figure 1. Total counted cells are indicated by green circles

Figure 2. Fluorescent image showing live Jurkat (red circles) and dead (green circles).

Cell and fluorescent size distribution histograms, scatter plots and data files can be instantly generated. All experimental data can be instantly transferred to an Excel spreadsheet or saved in a data table.

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