Evaluation of a short termin vitro test for granulocyte chalone activity

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Abstract
Summary A short termin vitro test for granulocyte chalone activity was examined for its specificity and reliability. The test uses the inhibition, by granulocyte extracts, of3H-thymidine (3H-Tdr) uptake into the acid-insoluble material by rat bone marrow cellsin vitro to measure possible chalone activity. Among the many possible3H-Tdr artifacts pool size dilution by Tdr contained in the extracts was excluded using an E. coli mutant requiring thymine. Several amino acids and biogenic amines do not affect the test. However, continuous and pulse labelling of bone marrow cells with3H-Tdr, viability tests and micro flow fluorometric measurements of the cell cycle distribution following colcemid treatment strongly suggest that the cells do not proliferatein vitro during short term incubation, since practically no cells enter the S-phase, cells in the S-phase die and few if any cells proceed through G2 and mitosis. Moreover, the test cannot exclude cytotoxicity. Thus, thein vitro test may only sceen for an unspecific S-phase inhibitor and must hence be supplemented by another assay to prove the chalone nature of an extract or fraction. The test per se fails to meet most of the requirements of a valid granulocyte chalone assay.