Flow Cytometry is a most popular analytical cell biology technique originally developed in the late 1960s which utilizes light to count and profile cells in a heterogeneous fluid mixture containing live cells. Flow Cytometry System is considered as the most powerful method because this system allows a particular researcher to accurately, rapidly and simply count the data and information related to many parameters by using light to count and also from a heterogeneous fluid mixture.
What Is Flow Cytometry System?
Flow cytometry is a powerful tool based on measurement of fluorescence associated with cells, which is typically used for immunology detection of monoclonal antibodies coupled to fluorochromes and it is also used to analyse multiple parameters on an individual cell basis. The flow cytometry system can be applied in any scenario and it is used extensively throughout the life and biomedical sciences where a researcher needs to rapidly profile and detect a large number of loose cells in a liquid media. The process in a flow cytometer includes:
- Different types of light are used in a flow cytometry experiment to illuminate the cells in the channel.
- Sample cells are passed through a tiny and narrow channel each at a time.
- A series of sensors detect the particular types of light that are emitted or refracted from the cells.
- Data received by the sensors is integrated and compiled to build a comprehensive picture of the sample.
The Cell Cycle Analysis by quantization of DNA was considered as one of the earliest applications of flow cytometry analysis and still continues to be one of the most important ones. In this process, the DNA of yeast, plant, mammal or any bacterial cells can be stained by a variety of DNA binding dyes and the main premise if this dye is that they are bind in proportion to the amount of DNA present in the particular cell. In flow cytometry, the total number of DNA per cell can be precisely determined to obtain cell cycle distributions.
Today, immunologists using various types of practical applications regularly include immunophenotyping, cellular proliferation, measuring intracellular cytokine production, cell cycle analysis and fluorescent proteins. Compared to all these applications, cell sorting based on flow cytometry technology is widely used to separate cells into populations of interest. There are various types of applications available for Flow Cytometry Immunology and it is very important to have the correct controls to set up the flow cytometer for any overlap in the emission of each of the fluorochromes. Boster antibody and ELISA experts are the reputable flow cytometry technical resource centre that provides all the protocols, optimisation tips related to flow cytometry in immunology.
For more details & information to know about Boster Antibody and ELISA experts please visit our website here: https://www.facs-analysis.com/.
Let’s look at the effective recommended for FACS Antibody:Harvest and clean cells and drag cell number to a concentration of 1-5x106 cells/ml in ice cold FACS Buffer.
But you can strain these in any container for which you have the accurate centrifuge such as eppendorf tubes, test tubes, and 96-well round-bottomed micro titer plates.
It is suggested to verify the viability of the cells which should be more than 95% and not less than 90%.
Moreover, it will be effective to do staining with ice cold solutions and at 4°C, because low temperature and presence of sodium azide prevent the modulation and internalization of surface antigens which could produce a loss of fluorescence intensity.Put 100 μl of cell suspension to every tube.The blocking antibody step is required however should be included if cells realizes high levels of Fc receptors.
Now incubate for at least half an hour at room temperature or 4°C in the dark.Clean the cells 3-4 times by centrifugation at 1500 rpm for ten minutes and then resuspend them in 200 μl to 1ml of ice cold FACS buffer.
Then clean the cells at least 5 times with the aid of centrifugation at 1500 rpm for 10 minutes and resuspend them in 200 μl to 1ml of ice-cold FACS buffer.
