Flow Cytometry Virtual Lab

Theoretical Background / Context

•  Flow cytometry is a laser-based technology that allows quantitative single-cell analysis. It is used to analyze the characteristics of cells.  
• Analysis of the cell cycle by DNA quantification can be done using a flow cytometry simulator. The DNA in cells can be stained by DNA binding dyes e.g. Propidium iodide. 
• These dyes bind in proportion to the amount of DNA present in the cell. 
• Cells in the S phase have more DNA than cells in the G1 phase. Cells in the G2 will have approximately twice the DNA content as cells in G1. Thus; they will take up proportionately more dye. 
• The suspension of cells is then aspirated into a flow cell.  Cells, surrounded by a narrow fluid stream, pass one by one through a focused laser beam. The light is either scattered or absorbed when it strikes a cell. 
• Absorbed light of the appropriate wavelength is reemitted as fluorescence. This reflects the internal structure of the cell and its size and shape. 
• Fluorescence scatter signals are detected, amplified, and analyzed by a series of photodiodes and a computer system.
• In the flow cytometry analysis, the end result is quantitative information about every cell analyzed. Large numbers of cells are analyzed in a short period of time (>1,000/sec). This gives the advantage of creating statistically valid information about cell populations.

The potential applications of flow cytometry technique include the detection and measurement of:

1. Cell cycle: Reliable assessment of cells in G0/G1 phase versus S phase, G2, or polyploidy, including analysis of cell proliferation and activation.
2. Cell viability/apoptosis.
3. Identification and characterization of distinct subsets of cells within a heterogeneous sample.
4. Protein expression.
5.  Protein post translational modifications.
6. RNA, including IncRNA, miRNA, and mRNA transcripts.  

Principle Work of Flow Cytometry Virtual Lab

• In flow cytometry virtual lab, cells are fixed and permeabilized using ethanol. Then, propidium iodide (PI) is used for staining DNA in intact cells.
• RNase is used to ensure only DNA is stained with PI. unstained cells are used as control.
• The end result of a flow cytometry virtual lab, is quantitative information about every cell analyzed. Large numbers of cells are analyzed in a short period of time (>1,000/sec).
• This gives the advantage of creating statistically valid information about cell populations.