Phenotyping suspended cells based on antigens present on the membrane is perhaps the most common use for flow cytometry. Because membrane proteins are readily accessible to the antibody, permeabilization steps are not required. However, experimental conditions, such as antibody concentration, incubation time, and temperature, should be optimized for each flow cytometry experiment. The following protocol has been developed and optimized by R&D Systems Flow Cytometry Laboratory for the staining of membrane-associated proteins.
Please read the protocol in its entirety before starting.
R&D Systems, Catalog # FC001, or an equivalent solution containing BSA and sodium azide
Note: Titration experiments should be performed to determine optimal reagent amounts.
Note: Do not wash excess blocking IgG from this reaction.
Note: If using whole blood, samples should go through a red blood cell lysis step at this point using Flow Cytometry Human or Mouse Lysis Buffer.
Note: If an unconjugated primary antibody was used, incubation with an appropriate secondary antibody should occur now. Dilute the secondary antibody in Flow Cytometry Staining Buffer, starting with the suggested concentration in the product datasheet. Incubate for 20-30 minutes in the dark and wash as in step 4.
Note: For a negative control, a separate set of cells should be stained with an isotype control antibody using the steps outlined above.
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|B220/CD45R Expression in Mouse Splenocytes. Mouse splenocytes were stained with an APC-conjugated Rat Anti-Mouse B220/CD45R Monoclonal Antibody (Catalog # FAB1217A; filled histogram) or an APC-conjugated Rat IgG2A Isotype Control (Catalog # IC006A; open histogram). B220 antigens represent a subset of mouse CD45 isoforms predominantly expressed on all B lymphocytes, including pro-, mature, and activated B cells. The B220/CD45 antigen antibody is commonly used as a B cell marker.|
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