LOSS OF PROTEINS AND OTHER MACROMOLECULES DURING PREPARATION OF CELL CULTURES FOR HIGH RESOLUTION AUTORADIOGRAPHY QUANTITATION BY A MICROMETHOD

A reproducible procedure was devised in order to measure the extraction of 3 H-labeled cellular products during aldehyde fixation and subsequent processing for electron microscopic autoradiography. Human carcinoma cell monolayers were cultivated in combustible plastic wells, so that all label could be counted as 3 H 2 O. Radioisotope extraction during individual steps of processing could be analyzed and separate groups of experiments were directly comparable. Initial aldehyde fixation and subsequent buffer washes caused the major loss of radiolabeled amino acids, but this never exceeded 15% under conventional conditions. Radioisotope losses were influenced by the relative duration of fixation and buffer washes, fixation temperature and fixative concentration. Formaldehyde and glutaraldehyde both produced a nonspecific, time-related binding effect when 3 H-labeled amino acids were introduced along with the fixative. Less significant nonspecific binding was observed when 3 H-mannose, 3 H-uridine or 3 H-thymidine was added. Extraction of radioisotopes during formaldehyde fixation of cell cultures labeled with protein precursors was consistently greater than during glutaraldehyde fixation. Differences were less marked with the other precursors. Evaluation of the total protein extraction is complex, since the net losses observed were apparently the sum of precursor extraction, nonspecific amino acid binding and real molecular extraction. The implications for quantitative interpretative interpretation of high resolution autoradiography must be considered.