Journal oflmmunologicalMethods, 64 (1983) 61-69 61 Elsevier JIM02802 Improved Culture Conditions for Quantitative Evaluation of Interleukin 2 Production by Frozen Human Lymphocytes Annick Harel-Bellan l,,, Carmen Marchiol 2 C6cile Kaplan 2 Jean-Yves Muller 2, Salem Chouaib 1.,, Arnaud Ythier l, Alexandre Nowill i and Didier Fradelizi l I INSERM U 93, H6pital Saint- Louis, 2, Place du Docteur Fournier, 75475 Paris 10, and 2 C.N. TS., 6, rue Alexandre Cabanel, 75015 Paris, France (Received 14 January 1983, accepted 20 May 1983) Several culture parameters were studied in order to establish methods for optimal and reproducible production of interleukin 2 (IL2) by thawed lymphocytes. Standard conditions, considered optimal for production by freshly separated lymphocytes (culture medium RPMI 1640 + 1% normal human serum + 10 /Lg/ml PHA), gave low and poorly reproducible results. An increased concentration of human serum (10 and 20%) in the medium improved production but best results were obtained by adding polyethylene glycol (PEG 6000, 0.1 mg/ml) to the culture medium. Furthermore, with the addition of PEG 6000, results became highly reproducible, thus permitting valid comparison of in vitro IL2 production by lymphocytes from normal donors and patients. Key words: interleukin 2 - polyethylene glycol thawed lymphocytes Introduction Interleukin 2 is a lymphokine produced by T lymphocytes stimulated by an antigen or mitogen. It stimulates the in vitro growth of activated helper and cytotoxic T cells amplifying the effector phase of immunity. The IL2 molecules (or family of molecules) may have a critical role in the development of an appropriate immune response in vivo. Altered IL2 production by lymphocytes of human individuals in the course of several diseases has been suspected (Wofsy et al., 1981). Reproducible methods for quantitative evaluation of IL2 production by human lymphocytes are required for measurement of this lymphokine in pathology. In fact, * A. Harel-Bellan and Salem Chouaib are supported by a fellowship from Sanofi Recherches Co. Abbreviations: IL2, interleukin 2; CM, culture medium; NHS, normal human serum; PBL, peripheral blood lymphocytes; PHA, phytohemagglutinin; PEG, polyethylene glycol. 0022-1759/83/5;03.00 © 1983 Elsevier Science Publishers B.V.