Hennessy, B and North, J and Deru, A and Llewellyn-Smith, N and Lowdell, MW (2001) Use of Leu3a/3b for the accurate determination of CD4 subsets for measurement of intracellular cytokines. CYTOMETRY , 44 (2) 148 - 152.
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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 sign-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. Cytometry 14:148-152, 2001. (C) 2001 Wiley-Liss, Inc.
|Title:||Use of Leu3a/3b for the accurate determination of CD4 subsets for measurement of intracellular cytokines|
|Keywords:||cytokines, CD4 subsets, T cells, human, FLOW-CYTOMETRY, CELLS|
|UCL classification:||UCL > School of Life and Medical Sciences > SLMS Planning and Performance Unit > SLMS Research Support Centre > Platform Technologies|
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