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. Moreover, never add sodium azide to buffers. Basically, cells for FACS Antibody general procedure are stained in polystyrene round-bottom 12 x 75 mm BD Falcon tubes. 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 put 100 μl of Fc block to every sample. Now incubate on ice for at least half an hour. Now centrifuge at 1500 rpm for 5-10 minutes at 4°C and finally discard supernatant.
Now put .1-10 μg/ml of the primary labeled antibody and do dilutions if required which must be made in flow cytometry buffer. 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. Moreover, store the cells in the dark on ice or at 4°C in a fridge in a case you need more time for analysis.
In a case, you use primary unlabeled antibody after completing step 4 do this- Dilute the fluorochrome-classified secondary antibody in FACS buffer at the finest dilution resuspend cells on this solution and incubate for as a minimum half an hour at room temperature or 4oC inside the darkish. 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. Lastly, store the cells in the dark on ice or at 4°C in a fridge till your scheduled time for analysis.
In a case, you need to store cells for many days, then after step 4, rather than re-suspending cells in 200 μl to 1ml of ice cold FACS buffer, put 100 μl 1-4% paraformaldehyde and incubate for 20 minutes at room temperature.
For effective effects of this General Experimental Procedure, examine the cells on the float cytometer as soon as viable. It is suggested analysis on the same day and in a case of extended storage (16 hr) and for higher flexibility in planning time at the Cytometry, resuspends cells in 1-4% paraformaldehyde to secure from deterioration.
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Flow Cytometry or FACS Analysis is not a new concept as it was developed back in 1960.
Well, people are keen to know that what types of light used in this process.And what is FACS Protocols.
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.
And, for this counting, so many types of lights used such as: Forward ScatterdMultiparametric AnalysisFluorescence EmissionSide Scatter What is FACS Staining Protocol?
It is available for direct and indirect staining of cells suitable where the fluorophore is straight linked to the primary antibody.
It is recommended not to use sodium azide to shields if you are involved with recovering cell function.
