Fixation and long‐term storage of human lymphocytes for surface marker analysis by flow cytometry

Abstract

A method to preserve stained human lymphocytes for subsequent cell surface analysis by flow cytometry (FCM) is described. Cells stained with fluorescein isothiocyanate (FITC) and phycoerythrin (€'E)-conjugated monoclonal antibodies and then fixed in 1% paraformaldehyde, followed by extensive washing and resuspension in 1% BSA medium, could be stored at 4°C for at least 2 weeks prior to FCM analysis without significant alteration in the light scatter or fluorescence properties of the cells. Furthermore, the method was also suitable for analyzing lymphocytes that express T-cell activation markers in certain disease conditions. In addition, we have identified monoclonal antibody combinations that discriminate different lymphocyte subsets that are satisfactory for multiparameter analysis after 2 weeks of storage. This method should prove useful for enumerating lymphocyte subsets in field study sites remote from flow cytometry laboratories.Key terms: Paraformaldehyde, filariasis, systemic lupus erythematosusThe lymphocyte population has been shown to be heterogeneous with respect to cell surface antigens (21). The functional properties of cells correlate strongly with these lineage-specific differentiation antigens (14). The subpopulations of B cells (1) and T cells (10) can be quantitated by flow cytometry (FCM) based on the relative expression of two or more cell-surface markers (8), and this approach has been used to define abnormal lymphocyte subsets in a variety of clinical conditions (9, 11).For such clinical studies, fresh lymphocytes (7) have generally had t o be used, since conflicting results have sometimes been observed with cryopreserved cells (31, an observation suggesting that certain differentiation antigens may be cryolabile. Thus, in most studies, cells are stained by conventional immunofluorescence techniques and analyzed either fresh or shortly after paraformaldehyde fixation (13). For studies carried out at sites remote from a flow cytometer, this protocol, however, creates practical problems, since the effect of longterm fixation on FCM analyses has not been well defined.Therefore the purpose of this study was to develop the optimal method of preserving stained cells for as long a time as possible. The basic requirement of such a technique would be that it permit 2-3 weeks storage of the cells prior to analysis without significant alteration of the light scatter and fluorescence properties of the cells. In this report, we describe such a method and define monoclonal antibody reagents satisfactory for studies requiring prolonged storage of FCM samples prior to analysis. MATERIALS AND METHODS Study PopulationThirteen individuals were studied. Eight were healthy North Americans, two were normal volunteers from New Delhi, India, two were patients with systemic lupus erythematosus, and one was an Indian patient with tropical pulmonary eosinophilia.Preparation of Fixative A 1.0% (w/v) solution of paraformaldehyde (Eastman Kodak Co., Rochester, NY) was prepared by dissolving paraformaldehyde in phosphat...

NOD-like receptors mediated activation of eosinophils interacting with bronchial epithelial cells: a link between innate immunity and allergic asthma

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