The fact is that FACS Protocols is used to examine different intracellular molecules comprising phosphorylated signaling proteins and cytokines. The Cytokines and other secreted molecules could be found while using the flow Cytometry in activated cells. Basically, these compounds save the export of synthesized proteins by providing the ER-Golgi transport machinery.

With the help of experimental treatments, the secretion inhibitor can be saved during the complete incubation period. In a case, the stimulation period is more than 5-6 hours; the secretion inhibitor must be combined for only the left hour 2 hours of the incubation.

Needless to say there are many variables required for individual flow Cytometry experiments. Moreover, to stain intracellular molecules, cells required to be fixed in suspension. Moreover, this fixation treatment endows the antibody to go through the plasma membrane into the cell interior, in fact, keeping the morphological characteristics used to find the cells.

Reagents Needed

Flow Cytometry Fixation Buffer

PBS (1X): 0.137 M NaCl, 0.05 M NaH2PO4, pH 7.4 or Hank’s Balanced Salt Solution (HBSS; 1X)

Isotype Control Antibodies

Flow Cytometry Permeabilization Buffer/Wash Buffer Detection Antibodies

Materials Needed

Pipette Tips and Pipettes

Vortex

FACS™ Tubes

Centrifuge

Procedure

Assemble the cells and clean 3 times with the aid of including 2 mL of PBS (or HBSS), centrifuging at three hundred x g for 5 mins, and then draining buffer from pelleted cell. It has to be cited that Intracellular Staining Protocol may be carried out at this factor.

Aliquot up to one x 106 cells/one hundred μL into FACS tubes. Assemble 0.5 mL of cold FAC Fixation Buffer and vortex. Incubate at room temperature for at least 15 mins. Vortex the cells intermittently on the way to preserve a single cell suspension.

Centrifuge cells and drain the Fixation Buffer. Then wash the cells 3-4 times with PBS (or HBSS).

Resuspend the cell pellet in 200 hundred μL of Flow Cytometry Permeabilization/Wash Buffer I.

Assemble 10 μL of the conjugated antibody and vortex. Incubate cells for half-hour at room temperature in the dark.

Wash cells 2 times with Flow Cytometry Permeabilization / Wash Buffer I. It should be noted that if an unconjugated primary antibody was used, incubation with an appropriate secondary antibody should occur now. Dilute the secondary antibody in Flow Cytometry Permeabilization / Wash Buffer I, starting with the concentration suggested in the product datasheet. Incubate for 20-30 minutes in the dark and wash.

Resuspend the cells in 300 – 500 μL PBS buffer for flow Cytometric analysis.

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