Use of Leu3a/3b for the accurate determination of CD4 subsets for measurement of intracellular cytokines.
BACKGROUND
Identification of human T-helper cell subsets is possible by measurement of intracellular cytokines after coincubation of lymphocytes with phorbol myristate acetate (PMA), calcium ionophore, and brefeldin A for up to 20 h. However, exposure to PMA leads to internalization of membrane CD4 and to loss of resolution of the CD4+ cells. Detection of CD3+CD8- cells or preselection of CD4+ cells prior to stimulation is more cumbersome than direct measurement of CD4+ cells. We report the use of the Leu3a/Leu3b multiclone for the accurate determination of CD4 cells after PMA stimulation.


METHODS
Peripheral blood lymphocytes were isolated from healthy normal donors and the proportion of CD3+ / CD4+ T cells was determined by flow cytometry before and after incubation with PMA, calcium ionophore, and brefeldin A for 20 h using a variety of anti-CD4 monoclonal antibodies.


RESULTS
The Leu3a/3b multiclone reagent was the only anti-CD4 monoclonal antibody capable of resolving more than 98% of the initial CD4+ events after incubation with PMA.


CONCLUSIONS
The higher signal-to-noise ratio associated with Leu3a3b reagent, compared with other CD4-specific antibodies available, allows the direct and accurate identification of the CD4 subset even after PMA treatment of cells.
Hennessy
B
B
Department of Haematology, Royal Free Campus, Royal Free and University College Medical School, London, United Kingdom.
North
J
J
Deru
A
A
Llewellyn-Smith
N
N
Lowdell
M W
MW
D015704:CD4 Antigens / Q000032:analysis / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007371:Interferon-gamma / Q000032:analysis*; D015847:Interleukin-4 / Q000032:analysis*; D007424:Intracellular Fluid
D015704:CD4 Antigens; D015847:Interleukin-4; D007371:Interferon-gamma