Use of Monoclonal Antibodies and Flow Cytometry to Cluster and Analyze Leukocyte Differentiation Molecules
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Recent advances in the design of flow cytometers and the development of associated software have facilitated the use of flow cytometry as a primary method for identifying and characterizing monoclonal antibodies (MAbs) reactive with molecules expressed on one or more lineages of leukocytes, erythrocytes, and/or platelets. It is now possible to distinguish mononuclear cells and granulocytes from erythrocytes and platelets on the basis of forward and right angle (orthogonal) light-scattering properties of cells using two parameter analysis (
1
–
3
; Chapter 15 ). It is possible to color-code the defined populations, and use single-color fluorescence analysis to determine whether a given MAb recognizes a molecule expressed on one or more lineages of leukocytes and/or platelets (Fig. 1 ) (
1
–
3
; Chapter 15 ). It is also possible to use two-color fluorescence analysis to simultaneously examine populations of resting and activated cells, and determine whether an MAb recognizes a molecule expressed on resting and/or activated cells (Fig. 2 ) (
4
–
6
). In this chapter, we describe the methods we have developed for using flow cytometry to screen primary tissue culture supernatants for the presence of MAbs specific for molecules expressed on resting and/or activated leukocytes. We also describe the use of multicolor fluorescence analysis to distinguish and cluster MAbs that identify determinants expressed on the same molecule or molecular complex.
Fig. 1.
Single-color analysis of whole preparation of leukocytes, using color gating to define granulocytes (red), monocytes (blue), lymphocytes (gold), and platelets (black).
Fig. 2.
Two-color analysis of the expression of membrane molecules on resting and Con A-activated lymphocytes using hydroethidine (a vital dye that is converted to ethidium in living cells) and MAb specific for CD3 or an activation molecule. The HE-loaded Con A blasts are clearly distinguished from resting lymphocytes in FL2. Cells labeled with MAbs specific for activation molecules or bovine CD3 are evident.
