Intracellular FACS Staining Protocol may be used to examine an expansion of intracellular molecules such as inflammatory mediators, transcription elements, cytokines, and phosphor proteins. It can ensure worthwhile information regarding cellular characteristic and signaling responses. Moreover, different from cell surface markers staining, intracellular antigens detection calls for cell fixation and permeabilization earlier than staining. Basically, cells are fixed with formaldehyde to hold the cellular morphology, after which permeabilized with alcohol or detergent. Such fixation treatment lets in the antibodies against intracellular antigens throughout the plasma membrane to stain intracellularly, whilst securing the morphological traits of cells.
It should be noted that for staining of secreted proteins, a transport inhibitor consisting of Brefeldin A or Monensin should be added prior to fixation a good way to entice the cytokines within the cells and enable intracellular staining.
Procedures:
Sample preparation: Assemble the tissues and cells and put together a single-cell suspension and modify the suspension to a concentration of 1 x 106cells/mL in cellular straining buffer. It should be noted that cell floor staining can be completed at this factor.
Fixation: Put 1 mL fixation solution in keeping with 1 x 106 cells and incubate for 20 minutes at room temperature. Then centrifuge at 400 x g for 5-8 minutes at room temperature and get rid of the fixation buffer. After this, clean fixed cells with cell staining buffer. Finally, centrifuge at 400 x g for 5-10 mins and discard the supernatant.
Permeabilization: In this step, resuspend constant cells in 2 mL permeabilization buffer and centrifuge at 400 x g for 5-7 minutes at room temperature, then remove the supernatant. After this, clean the permeabilized cells with permeabilization buffer, then Centrifuge at 400 x g for 5 minutes and drop the supernatant. Once done, repeat this step again.
Staining: Mix the primary antibody with permeabilization for a foremost operating concentration and resuspend the permeabilized cells with the primary antibody solution. Then incubate for 20 minutes at 4°C. It should be noted that in a case, the use of primary antibodies without delay conjugated with the fluorochrome, and then incubation must be done t within the dark. Then centrifuge at 400 x g for 5 minutes at 4°C and drop the supernatant. Finally, clean with permeabilization buffer and centrifuge at 350 x g for 8 minutes and drop the supernatant. Once done, repeat this step again.
After this, demise the fluorescent-conjugated secondary antibody with permeabilization buffer for a most reliable running concentration and resuspend the cell pellet with the secondary antibody solution. Then, incubate on ice for 20-25 minutes in the dark. Once done with it, at least 3 times wash with permeabilization buffer and centrifuge at 350 x g for 5-8 mins. Discard the supernatant.
Resuspend cells in 300-500 uL cellular staining buffer for final flow Cytometry analysis.
If you have any question regarding Intracellular Staining Protocol, please contact Boster Antibody & ELISA at https://www.facs-analysis.com/. It has a veteran team that can guide the entire procedure comprehensively.
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.
