Use of Leu3a/3b for the accurate determination of CD4 subsets for measurement of intracellular cytokines

✍ Scribed by Brian Hennessy; Janet North; Akinwale Deru; Nigel Llewellyn-Smith; Mark W. Lowdell

• Publisher: John Wiley and Sons
• Year: 2001
• Tongue: English
• Weight: 192 KB
• Volume: 44
• Category: Article
• ISSN: 0196-4763
• DOI: 10.1002/1097-0320(20010601)44:2<148::aid-cyto1094>3.0.co;2-6

✦ Synopsis

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.