Quantitative cytochemical analysis of (single) cultured cells

Human fibroblasts or amniotic fluid cells can be cultivated in special dishes with a bottom of thin transparent plastic foil. After quick freezing and freeze-drying small groups of cultured cells or single cells can be isolated by dissecting small pieces of plastic foil under the stereomicroscope. These can be incubated in (sub)microlitre volumes of substrate and the absorbance, fluorescence or radioactivity can be measured and expressed per cell. When absorbance is measured with a microscope spectrophotometer in 1-10 microliter, microcuvettes, the minimum amount of coloured product that can be determined is of the order of 10(-11) moles. Incubation under paraffin oil in 0.05 microliter of substrate, followed by fluorescence measurements in 1-5 microliter with a microscope fluorometer, allows down to 10(-14) mol of methylumbelliferone, which is the end-product in many analyses of lysosomal enzymes, to be detected. (Re)cycling procedures have an even greater sensitivity (10(-15)-10(-19)mol) but they are more complicated and limited to NAD(P)(H)-dependent reactions. Several radiometric procedures have also been adapted for the analysis of small numbers of cultured cells but most of them still require 10(4)-10(5) human fibroblasts. These microtechniques allow rapid prenatal diagnosis of at least 20 different genetic metabolic diseases to be made within 7-14 days after amniocentesis at the 14th-16th week of pregnancy. Enzyme assays on single cultured cells have also enabled complementation studies to be done on heterokaryons after hybridization of different types of mutant fibroblasts, as well as investigations of the intercellular exchange of lysosomal enzymes between normal and mutant cells.