Identification and quantification of human peripheral blood lymphocytes and monocytes following migration into...

Previously, in order to evaluate the migration of specific types of lymphocytes (e.g., T or B cells, CD4 or CD8 subsets) using the Boyden chamber technique, the relevant cell populations have first been purified. We report here a method which permits identification of the lymphocytes following migration into the nitrocellulose filters. After fixation, the filters are exposed to specific anti-lymphocyte monoclonal antibodies and the reaction is visualized via a second gold-linked antibody. Monocyte-depleted lymphocytes isolated from human peripheral blood are routinely used for migration but mixed mononuclear cell preparations can also be used. This was shown by comparing lymphocyte migration in monocyte-containing and monocyte-depleted cell suspensions isolated from the same donor. The presence of monocytes did not influence the migration of normal resting T lymphocytes. With human peripheral blood as starting material the method is most suitable for the evaluation of T cell migration since, in most instances, T cells are the predominant lymphocyte population. When the B cell count is normal (5-10% total lymphocytes), quantification of B cell migration requires enrichment but not complete purification of this population. Migrating monocytes can also be identified. However, the antibody staining is less intense on the spread monocytes and, therefore, quantification must be performed by eye rather than with the Optomax image analyser.