Hi we are examining CD81 expression in B cells, macrophages and neutrophils in PBL from mice. To this end, we collected 10 ul blood, lysed RBC, and stained in 10 ul antibody cocktail (B220, CD81, Ly6G, CD11b).
The CD81 staining was stable on B cells , but not on mac and Neut where the intensity varied up to 10x for the same mice in sequential experiments using the same prep of antibody cocktail. However, anti-Ly6G, which marks Neut, yielded consistent signals (as were CD11b and B220) and so the variability of CD81 is specific to CD81. Any advice? BTW, anti-CD81 (EAT2) is from Biolegend. Thx.
Hi Tian
CD81, along with other tetraspanins, have differential expression on subsets of myeloid cells and can be influenced by various conditions such as inflammation.
This article may be useful to you:
Tippett E, Cameron PU, Marsh M, Crowe SM. Characterization of tetraspanins CD9, CD53, CD63, and CD81 in monocytes and macrophages in HIV-1 infection. J Leukoc Biol. 2013;93(6):913-20.
Tetraspanins are a family of membrane-organizing proteins that mediate diverse functions. Little is known of their expression or function in myeloid cells. Here, expression of CD9, CD53, CD63, and CD81, tetraspanins that have been implicated in HIV-1 pathogenesis, were characterized in normal monocyte subsets, in MDM, and in HIV-1-infected donors. We show that tetraspanins are expressed differentially by monocyte subsets, with higher CD9 and CD63 and lower CD53 and CD81 levels on CD14++CD16- monocytes compared with CD14++CD16+ and CD14+CD16++ subsets. Maturation of monocytes resulted in increased CD9 expression and apparent relocation of CD63 and CD53 from surface to intracellular membranes. Expression was modulated by cytokines, and CD9 was a marker of anti-inflammatory and CD53 a marker of proinflammatory MDM. Tetraspanin expression on monocyte subsets from HIV-1-infected donors receiving antiretroviral therapy was unchanged compared with that in uninfected donors. However, CD53 expression was inversely correlated with viral load in HIV-1-infected donors not on therapy. This study is the first to comprehensively characterize tetraspanin expression on monocyte subsets and macrophages in health and during HIV-1 infection. It demonstrates regulation of tetraspanin expression by cytokines, and CD53 expression as a novel correlate of a proinflammatory phenotype. This paper characterizes tetraspanins in myeloid cells and shows that tetraspanins are expressed differentially in monocyte subsets and are modified in inflammatory conditions.
Regards, Rosemary
http://www.ncbi.nlm.nih.gov/pubmed/23570947
Hi,
As far as i know, neutrophils do not express CD81. Fluorescence dispersion should be explained because of the low proportion of these cells in blood and the sample volume, so you're not getting enough events to make a representative sampling with normal distribution.
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