Ž . Journal of Immunological Methods 258 2001 193–198 www.elsevier.comrlocaterjim Protocol Intracellular cytokine staining for TGF-b Mohammed L. Garba, Jeffrey A. Frelinger ) Department of Microbiology and Immunology, UniÕersity of North Carolina, CBa7290 MEJ, Chapel Hill, NC 27599-7290, USA Received 7 August 2001; accepted 22 August 2001 Abstract TGF-b is a well-described immunoregulatory molecule that is produced by most cell types. Many studies have been aimed at investigating the role played by TGF-b in different cell types and situations. Most methods of measuring TGF-b Ž . have previously relied on enzyme-linked immunosorbent assay ELISA or assays of its anti-proliferative effects on various cell lines. Both assays, though useful, cannot be used to effectively identify the cells that are producing TGF-b in a mixture of cells. It is especially important to know the source and dynamics of TGF-b secretion in cell culture studies since most cell types are known to be capable of producing TGF-b. We describe here a technique of qualitative and quantitative measurement of TGF-b production using flow cytometry. Previous work by others has led to the production of polyclonal and monoclonal antibodies to human and other mammalian TGF-b. We have developed an intracellular cytokine staining for human TGF-b using a monoclonal antibody, TB21. q 2001 Elsevier Science B.V. All rights reserved. Keywords: TGF-b; Flow cytometry; Intracellular; T cells; Cell line 1. Background Ž . Transforming growth factor-beta TGF-b is a 25-kDa nonglycosylated homodimer protein consist- ing of two 12.5-kDa subunits linked by disulfide Ž . bonds Lawrence, 1996 . It is a part of the super family containing TGF-a , bone morphogenic pro- Ž . teins, activins and inhibins Cheifetz et al., 1987 . It Ž . consists of five subunits TGF-b1– b5 and is highly Ž conserved across mammalian species Alevizopoulos . and Mermod, 1997 . TGF-b is broadly immunosuppressive. It was ini- tially described and named based on its ability to ) Corresponding author. Tel.: q 1-919-966-2599; fax: q 1-919- 962-8103. Ž . E-mail address: jfrelin@med.unc.edu J.A. Frelinger . transform fibroblasts into a tumor-like phenotype Ž . Lawrence, 1996 . It is most commonly believed to be an important modulator of immune functions Ž including cell growth and differentiation Sporn and Roberts, 1990; Su et al., 1991; Vogelbruch et al., . 1995; Letterio and Roberts, 1998 . TGF-b is classi- fied as a Th3 cytokine and can be produced by most Ž mammalian cells Alevizopoulos and Mermod, 1997; . Cerwenka and Swain, 1999 . TGF-b receptors are also found on most mammalian cells. The most abundant types of TGF-b are the TGF- Ž b1 and -b 2, which are 70% homologous Cheifetz et . al., 1987 . TGF-b1 is believed to be responsible for Ž most of the inhibitory effects of TGF-b Ohta et al., . 1987 . It is formed from the cleavage of pro-TGF-b1 at its C-terminus by a protease and secreted by the cells in an inactive or latent form bound to latency Ž . associated proteins LAP and the latent TGF-b 0022-1759r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. Ž . PII: S0022-1759 01 00491-4