Detergent treatment of dictyostelium discoideum cells allows examination of internal cell type‐specific antigens by flow cytometry

Abstract

Monoclonal antibodies are used extensively in flow cytometry to identify subpopulations of cells differing in surface antigens. Conventional studies on living cells do not allow analysis of internal antigens, because antibody molecules do not pass through an intact plasma membrane. It is important for developmental studies on Dictgostelium discoideum that not only surface but also internal antigens be analysed. Here techniques are reported that make possible such studies by permeabilising cells with mild detergent treatments using digitonin. Over the past ten years there has been a revolution in culture cells that it is possible to permeabilise them so the understanding of the differentiation of lymphocyte that antibodies have access to internal antigens for flow subpopulations. The isolation of monoclonal antibodies cytometric measurements (14). that recognise antigenic determinants exposed at the In this report, studies that optimise techniques for surface of blood cell subpopulations and their applica-analysing internal antigens of D. discoideurn slug cells tion in flow cytometry has hastened this understanding are described. With mild detergent treatments, it is pos-(15). We have developed techniques for disaggregating sible to analyse internal antigens of unfixed cells. The the cells of the cellular slime mold Dictyostelium discoz-techniques outlined here are simple and rapid. We use deum slug, which is one of the simplest multicellular digitonin to permeabilise unfixed cells and to demoneukaryotes, and analysing them by flow cytometry (16). strate the effectiveness of this technique using two preOur studies centre on the differentiation of this organ-spore-specific internal antigens. ism, to produce two predominant cell types, the prespore and prestalk cells (16). In this way, cell surface antigens MATERIALS AND METHODS that identify prespore (9) or prestalk (10) cells have been Materials discovered.Digitonin (80% pure) was from Calbiochem. DeterIn recent work antigens that are cell type-specific but gents Triton X-100 and Nonidet P-40 were from Sigma. not found at the cell surface have been discovered (Alex-Monoclonal antibodies MUD1 (71, MUD3 (171, and ander et al., submitted). While internal antigens can be MUD102 (Smith, unpublished) raised against D. discoistudied after sorting the cells on the basis of cell surface markers, it would be more convenient to be able to detect internal antigens, directly in mixed-cell popula-'This research was supported by a NH & MRC (Australia) p a n t to tions, in the Same way that cell surface antigens are KLW. RLB was on sabbatical leave from San Francisco State studied by flow cytometry. Since antibodies cannot pen-u~~~~~r e p r i n t requests to Keith Williams, School of Biological etrate intact flow cytometry precludes such studies. However, it has been shown in tissue 2109.

Immunodominant carbohydrate determinants in the multicellular stages of Dictyostelium discoideum

“…Detection was on a Coulter Electronics EPICS V flow cytometer, with an argon-ion laser operating at 488 nm. Methods of sample preparation and the range of machine settings have been reported previously (Alexander et al, 1988Bernstein et al, 1988. Competition assays between mAbs MUD50 and MUDl 15, and MUD122 and MUD124 were performed by preincubation of disaggregated slug cells with excess unlabelled antibody (180 pl cell culture supernatant/:!…”

“…They also behaved similarly to MUD62 in flow cytometry in that they did not bind to the surface of prestalk or prespore cells, but labelled the surface of mature spores (data not shown). MUD62 has been shown to identify internal antigens in prespore cells when digitonin is used to permeabilize slug cells (Bernstein et al, 1988). The remaining mAbs identified at screening showed three different patterns, two of which will be discussed elsewhere.…”

A monoclonal antibody that interferes with the post‐aggregation adhesion of Dictyostelium discoideum cells

“…WGA-purified 90 kDa antigen and PsB co-migrate on gels, however, PsB is thought to be a soluble internal pre-spore protein [34,44] whereas the 90 kDa antigen is present in crude plasma membrane fractions and both spores and stalk cells. The 35 kDa antigen is stalk-specific and probably the small st35 protein identified by others [45].…”

Gradients in the expression of cell surface glycoproteins in a simple tissue, the Dictyostelium discoideum slug

“…For single-colour analysis each cell was analysed for two variables : forward angle light scatter (FALS) as a measure of relative size, and integrated green fluorescence as a measure of number of mAb molecules attached. Two-colour analysis was as described in Bernstein et al (1988).…”