Cancer Cell Research

Apoptosis assays are routinely used in many laboratories for variety purposes from assessing the health of cell cultures to evaluating the toxicity of a panel of compounds.
Apoptosis assay is a type used for determining the apoptosis percentage of cells by Annexin V-FITC/PI staining method. Annexin V binds to phosphatidylserine (PS) with early apoptosis cell or necrosis cell. PI only enters necrotic/very late-stage apoptotic cells. (Figure 1)
A： Early apoptosis Annexin V (+),  PI (-)
B：Late apoptosis Annexin V (+), PI (+)


Figure1: Enlarged details of Countstar Rigel pictures (5 x magnification) of 293 cells, treated with Annexin V FITC and PI

The cell cycle or cell-division cycle is the series of events that take place in a cell leading to its division and duplication of its DNA (DNA replication) to produce two daughter cells. In cells with a nucleus, as in eukaryotes, the cell cycle is also divided into three periods: interphase, the mitotic (M) phase, and cytokinesis. Propidium iodide (PI) is a nuclear staining dye that is frequently used to measure cell cycle. Because the dye cannot enter live cells, the cells are fixed with ethanol prior to staining. All of the cells are then stained. Cells preparing for division will contain increasing amounts of DNA and display proportionally increased fluorescence. Differences in fluorescence intensity are used to determine the percentage of cells in each phase of the cell cycle. Countstar can capture the image and results will be displayed in FCS express software. (Figure 2)


Figure 2: MCF-7 (A) and 293T (B) were stained with cell cycle Detection Kit with PI, the results were determined by Countstar Rigel, and analyzed by FCS express.

During the bioprocess, GFP is often used to fuse with recombinant protein as an indicator. Determine the GFP fluorescent can reflected the target protein expression. Countstar Rigel offers a fast and simple assay for testing GFP transfection as well as viability. Cells were stained with Propidium iodide (PI) and Hoechst 33342 to define the dead cell population and total cell population. Countstar Rigel offers a quick, quantitative method for evaluating GFP expression efficiency and viability at the same time. (Figure 4)


Figure 4: Cells are located using Hoechst 33342 (blue) and the percentage of GFP expressing cells (green) can easily be determined. Nonviable cell are stained with propidium iodide (PI; red).

AO/PI Dual-fluoresces counting is the assay type used for detecting cell concentration, viability. It divided into cell line counting and primary cell counting according to different cell type. The solution contains a combination of the green-fluorescent nucleic acid stain, acridine orange, and the redfluorescent nucleic acid stain, propidium iodide. Propidium iodide is a membrane exclusion dye that only enters cells with compromised membranes while acridine orange penetrates all cells in a population. When both dyes are present in the nucleus, propidium iodide causes a reduction in acridine orange fluorescence by fluorescence resonance energy transfer (FRET). As a result, nucleated cells with intact membranes stain fluorescent green and are counted as live, whereas nucleated cells with compromised membranes only stain fluorescent red and are counted as dead when using the Countstar Rigel system. Non-nucleated material such as red blood cells, platelets and debris do not fluoresce and are ignored by the Countstar Rigel software. (Figure 5)


Figure 5: Countstar has optimized a dual-fluorescence staining method for simple, accurate determination of PBMC concentration and viability. Samples stained with AO/PI can be analyzed with the Counstar Rigel