Ten-color immunophenotyping of hematopoietic cells

Multicolor flow cytometry offers the unique ability to simultaneously assess and correlate multiple cellular properties at the single-cell level in a timely and efficient manner. Principles necessary for the development and evaluation of 10-color flow cytometry panels are discussed. The Basic Protocol outlines a simple and efficient method for the labeling of white blood cells with monoclonal antibodies directed against cell surface antigens. Alternate Protocol 1 incorporates the removal of plasma to allow the simultaneous assessment of surface light-chain expression on B cell populations. Alternate Protocol 2 describes a general method for the simultaneous assessment of surface and cytoplasmic antigens using a combination of fixation followed by membrane permeabilization. The methods were developed in a clinical laboratory setting for the description of normal pathways of hematopoietic maturation and the efficient identification of neoplastic hematopoietic cell populations, but the general principles should also be suitable for other applications.