Immunophenotyping tip sheet

Understanding Fc blocking

Intact antibodies will bind to any cell that has an unoccupied Fc receptor. Only thymocytes and erythrocytes are void of Fc receptors on their cell surface. It is important to use an Fc block to bind up the Fc sites prior to incubation with antibodies. CD16/32 specifically binds to Fc receptors and can actually be used in conjunction with a second antibody to identify Fc receptor-carrying cells.

Understanding dead cells

Dead cells have the ability to pick up free-floating fluorescence dyes. By using a dead cell marker such as propidium Iodide, 7-AAD or DAPI, cells that have a permeabilized nuclear membrane can absorb these dyes, which bind to nucleic acids. These dyes fluoresce and can be subsequently excluded from data acquisition via live cell gating.

Understanding binding

There are two kinds of binding: specific and non-specific. Specific activity includes Fab to epitope and Fc to Fc Receptor. Non-specific is an across-the-board low-affinity stain that is non-saturable. Antibodies do not bind due to over-conjugation, non-purification, binding site degradation or aggregation.

Understanding antibody dilutions

Most antibodies purchased from companies arrive with suggested working dilutions. Perform a titration on these antibodies to ensure they work and to determine the stain index. This ensures you are using the optimal dilution to obtain the best resolution between the positive and negative fraction.