Have a Brief Guide on Flow Cytometry Antibody Staining Procedure

Harvest and clean the cells and regulate cell number to a concentration of 1-5x106 cells/ml in ice-cold FACS Antibody Buffer. It should be noted that no need to add sodium azide to buffers if you are worried about getting better cell function. The cells are generally stained in polystyrene spherical-bottom 12 x 75 mm BD Falcon tubes. But they can be stained in any field for which you have the ideal centrifuge. It is constantly useful to test the viability of the cells which ought to be around ninety-five percentage but now not less than ninety per cent. It is advisable to stain with ice-cold solutions and at 5°C because low temperature and presence of sodium azide save you the modulation and internalization of surface antigens that can provide a loss of fluorescence depth.

Put 100?l of cell suspension to every tube.

To perform step 3 is optional but it can be done. If cells express excessive tiers of Fc receptors with the intention to make contributions to non-specific binding and background fluorescence, then put a hundred?l of Fc block to every sample. Then Incubate on ice for 25 minutes. Centrifuge at 1500 rpm for 5-8 min at four°C. Finally, throw out supernatant.

In step 4, put 0.1-10?g/ml of the primary labelled antibody. In a case, dilutions are necessary then need to be made in FACS buffer. Then, incubate for at the least 30-40 min at room temperature or 4°C within the darkish. Moreover, this step will need optimization.

Wash the cells 3-4 times by means of centrifugation at 1500 rpm for 5-10 mins and resuspend them in 200?l to 1ml of ice-cold FACS buffer. Make sure to keep the cells within the dark on ice or at 4°C in a fridge until your scheduled time for evaluation. If you put in use the primary unlabeled antibody after completing the above step then dilute the fluorochrome-classified secondary antibody in FACS buffer on the optimal dilution, resuspend cells in this solution and incubate for at least 30 minutes at room temperature within the darkish. After this, clean the cells 3-4 times by means of centrifugation at 1500 rpm for 5-8 minutes and resuspend them in 200?l to 1ml of ice bloodless FACS buffer. Keep the cells within the dark on ice or at 4°C in a fridge until your scheduled time for evaluation.

If you need to maintain cells for some days, infectious materials or microorganism, after finishing step 4 as opposed to resuspending cells in 200?l to 1ml of ice-cold FACS buffer, upload 100?l 1-4% paraformaldehyde and incubate for 15-20 min at room temperature. Then centrifuge your samples at 1500 rpm for five min and resuspend them in 2 hundred?l to 1 ml of ice-cold PBS. Moreover, fixation will disable most biohazardous agents, reduce deterioration and assist to maintain the integrity of your samples. The amount of fixative wished for one-of-a-kind sample kinds will require optimization by way of the consumer.

Analysis:

For best results of this General Experimental procedure, make sure to analyze the cells on the flow Cytometry as soon as possible. It is recommended to complete this procedure on the same day.

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