Detergent treatment of Dictyostelium discoideum cells allows examination of internal cell type-specific antigens by flow cytometry.
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 Dictyostelium 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. Flow cytometer profiles of unfixed cells show that antigens recognised by two monoclonal antibodies, MUD102 and MUD3, are found inside subpopulations of cells in the D. discoideum slug. Double-labelling experiments were carried out to demonstrate that the antigens recognised by these antibodies are present inside prespore but not prestalk cells. The detergent treatment leads to loss of forward-angle light scatter, but 90 degrees light scatter of cells is not greatly affected. While fixed cells sometimes gave satisfactory results, internal labelling did not reliably demonstrate the two subpopulations observed with unfixed cells.
Bernstein
R L
RL
School of Biological Sciences, Macquarie University, Sydney, New South Wales, Australia.
Browne
L H
LH
Yu
S C
SC
Williams
K L
KL
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D004023:Dictyostelium / Q000166:cytology* / Q000187:drug effects / Q000276:immunology; D004072:Digitonin / Q000494:pharmacology*; D000939:Epitopes / Q000032:analysis*; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique; D008722:Methods; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011092:Polyethylene Glycols; D017830:Octoxynol; D004072:Digitonin