FACS Protocol for Staining Intracellular Molecules Using Detergents to Permeabilize the Cell Membrane 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 NaH 2PO4, pH 7.4 or Hank’s Balanced Salt Solution (HBSS; 1X) Isotype Control Antibodies Flow Cytometry Permeabilization Buffer/Wash Buffer Detection Antibodies