Let’s find out FACS Antibody Staining Procedure:
In the beginning, harvest and clean the cell and then adjust cell number to a concentration of 1-5x106 cells/ml in the ice-cold FACS Buffer. It should be noted that never put sodium azide to buffers if you are required with recovering cell function. For instance, if cells are to be assembled for functional assays, then it should inhibit metabolic activity. In addition, cells are basically stained in polystyrene round-bottom 12 x 75 mm BD Falcon tubes but you can strain these in any container for which you have an exact centrifuge. For instance - Eppendorf tubes, test tubes, and 96-well round-bottomed micro titer plates. These are useful to analyze the viability of the cells which must be approximately 95% but not less than 90%.
Moreover, it is suggested that staining should do with ice-cold solutions and at 4°C since low temperature and presence of sodium azide save the modulation and internalization of surface antigens which can result in a loss of fluorescence intensity.
Now add or put 100 μl of cell suspension to every tube.
Step 3 is essential for blocking antibody however should be included in a case if cells express high levels of Fc receptors which will distribute to non-specific binding and background fluorescence. In the 3 steps of General Experimental Procedure, make sure to put 100 μl of Fc block to each sample then incubate on ice for half an hour, then centrifuge at 1500 rpm for 5-10 min at 4°C and finally discard supernatant.
Put or add 0.1-10 μg/ml of the primary labelled antibody.
Now incubate for at least half an hour at room temperature or 4°C in the dark. Moreover, this step needs optimization.
Mow clean cells for at least 3-4 times by centrifugation at 1500 rpm for 10 minutes and resuspend them in 200 μl to 1ml of ice-cold FACS buffer. Make sure to store the cells in the dark on ice or at 4°C in a fridge until your scheduled time for analysis. In a case, you will use primary unlabeled antibody once accomplished step 5, then do the following steps:
Dilute the fluorochrome-labelled secondary antibody in Flow Cytometry buffer at the maximum dilution, resuspend cells in this solution and incubate for minimum half an hour at room temperature or 4oC in the dark.
Clean the cells 3-4 times by centrifugation at 1500 rpm for 10 minutes and resuspend them in 200 μl to 1ml of ice-cold FACS buffer. Moreover, make sure to store the cells in the dark on ice or at 4°C in a fridge until your scheduled time for analysis.
In a case, you need to store cells for many days once accomplished step 5 instead of resuspending cells in 200 μl to 1ml of ice-cold FACS buffer, put100 μl 1-4% paraformaldehyde and incubated for just 15-20 minutes at room temperature.
Now, centrifuge your samples at 1500 rpm for 10 minutes and resuspend them in 200 μl to 1 ml of ice-cold PBS. Moreover, MoFixation will inactivate most biohazardous agents, decline deterioration and assist to preserve the integrity of your samples.
For best results, make sure to analyze the cells on the flow cytometer as soon as possible. For this, Booster antibody and ELISA experts suggest analysis on the same day. In a case of extended storage and greater flexibility in planning time on the cytometer, resuspend cells in 1-4% paraformaldehyde to get away from deterioration.
Booster antibody and ELISA experts is an eminent destination to experience the complete, simple, and accurate Flow Cytometry Antibody Staining Procedure. To get more help, get in touch with its official website https://www.facs-analysis.com/.