Each human cell expresses hundreds of thousands of cell surface antigens that specify their cell type, biological function, development stage, and more. Cells from different organs have characteristic surface antigens, and we can
utilize fluorochrome-conjugated antibodies specific for these surface markers to analyze these cells directly by flow cytometry. In direct immunofluorescence staining, cells are incubated with an antibody directly conjugated to a
fluorophore such as PerCP. This requires only one antibody incubation step and therefore eliminates the possibility of non-specific binding from a secondary antibody. Direct staining is advantageous during intracellular staining because
large antibody-fluorophore complexes including secondary antibodies can become trapped and result in non-specific binding, or they may fail to enter the cell which results in no detection.
If the experiment will be staining with unconjugated purified antibody, there needs to be an additional step of staining with a fluorescent conjugated secondary antibody (indirect staining).
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In indirect staining, the fluorophore conjugated secondary antibody detects the primary antibody which is unconjugated. Another available method is the avidin-biotin system, whereby a biotin-conjugated antibody is detected with
fluorophore-labeled avidin. There is a wide range of conjugated antibodies available today, and with indirect staining, the choices of target proteins further increases. Now unconjugated primary antibodies raised against many different
targets can be used together with a conjugated secondary antibody for flow cytometric analysis.
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In addition to the cell surface antigens, the intracellular staining procedure allows direct measurement of antigens (cytokines or transcription factors) present inside the cell cytoplasm or nucleus. In this procedure, the fixation and
permeabilization of cells are required. Fixing cells will retain the target protein in its original cellular location, and will usually ensure better stability of soluble antigens and antigens with a short half-life. Detecting
intracellular antigens requires cell permeabilization before staining, and antibodies should be prepared in permeabilization buffer to ensure the cells remain permeable. This modified staining procedure allows direct measurement of
functional activity of any cell of interest present in blood or other tissues without further separation.
Various fixation and permeabilization methods are available to allow access of antibodies to intracellular proteins. To achieve better results, additional in vitro stimulation with some common mitogen may be required to trigger increased
production of cytokines inside cells. Some common mitogens for this purpose include PMA, Ca++ or peptide epitopes and protein transport inhibitor, Brefeldin A, etc. Detecting secreted proteins can be difficult if the proteins are released
from the cell before detection, or if they degrade rapidly. In these situations, we recommend using Brefeldin A or other compounds that prevent protein release from the Golgi apparatus. A Golgi block such as Brefeldin A will inhibit
secretion of expressed proteins from the Golgi apparatus, trapping them in the cell to be detected. The intracellular staining method can then be utilized for detection of the target protein.
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