Flow Cytometry or FACS Analysis is not a new concept as it was developed back in 1960. It is actually the analytical cell-biology system that uses light to count and profile cells in a heterogeneous fluid mixture. Well, people are keen to know that what types of light used in this process.And what is FACS Protocols. So, today in this article we will discuss the same but, first, you should know what actually the flow Cytometry is.
What is Flow Cytometry?
It is one of the most important inventions of the 1960s as it allows researchers to get or collect data accurately and rapidly to many parameters from a heterogeneous fluid mixture which has live cell included. It is the part of the biomedical science and used in it for various purpose.
Types of light used in a flow Cytometry experiment are as follows:
In FACS low cytometer does emit light to count numbers of the cell. And, for this counting, so many types of lights used such as:
- Forward Scatterd
- Multiparametric Analysis
- Fluorescence Emission
- Side Scatter
What is FACS Staining Protocol?
FACS Staining Protocol is the indirect staining of intracellular antigens, permeabilization and cell preparation protocols. It is available for direct and indirect staining of cells suitable where the fluorophore is straight linked to the primary antibody. It is also applicable for preparation of cell as well as DNA staining for cell.
Here are the ways of FACS Staining Protocol:
- Wash the cells then, adjust cell amount. It is recommended not to use sodium azide to shields if you are involved with recovering cell function. Intracellular Staining Protocol is used for stained cells in polystyrene round-bottom.
- In each tube add 100 μl of cell suspension.
- If the cells express high levels of Fc receptors then, add 100 μl of Fc block in every sample and, nurture it in an ice for 20 min.
- The 0.1-10 μg/ml of the primary labelled antibody need to be added after the previous way.
- Now, incubate at room temperature for at least 30 min. Optimization is required in this step.
- For 5 minutes wash the cells at least 3 times by centrifugation at 1500 rpm. When you have done then, resuspend in 200 μl to 1ml of ice-cold FACS buffer and incubate it is the dark or for at least 4°C in a fridge.
- After completing step 5, if you want to preserve the cells of human and animal then, do not resuspend cells in 200 μl to 1ml of ice-cold FACS buffer. In that place just add 100 μl 1-4% paraformaldehyde and incubate for 10-15 min at room temperature.
- It is good to analysis the flow cytometer for best results as soon as possible. Well, the ideal time to analysis the cells is the same day when you keep it to preserve. Well, for greater flexibility and extended storage resuspend cells to prevent deterioration in 1-4% paraformaldehyde.
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.
