A SIMPLE MICRO-ASSAY FOR ERYTHROPOIETIN BASED ON THYMIDINE-H-3 INCORPORATION INTO SPLEEN-CELLS FROM PHENYLHYDRAZINE TREATED MICE

  • 1 January 1983
    • journal article
    • research article
    • Vol. 11  (7) , 649-660
Abstract
Spleens taken from B6C3F1 mice on the 3rd day following the 2nd of 2 daily injections of phenylhydrazine HCl (60 mg/kg) contain .apprx. 10 times the number of cells (i.e., 109 cells) found in spleens from normal mice. Of these spleen cells, > 90% are recognizably erythroid and form the basis of a new in vitro microassay for erythropoietin (Ep) which uses 3H-thymidine incorporation as an endpoint. This assay takes 24 h and can be carried out in the presence of either 20% FCS [fetal calf serum] or a well-defined serum substitute. Under the conditions used, Ep levels from 30 mU/well down to as little as 0.2 mU/well can be accurately measured with a corresponding variation in counts of 50- to 100-fold. One spleen is sufficient for 1000 triplicate Ep determinations, and the use of this microassay procedure requires only very small (1-60 .mu.l) samples for evaluation. Preliminary studies with human plasma suggest that this assay may be more specific for Ep than established in vitro 59Fe bioassay methods.