A simple microassay for erythropoietin based on 3H-thymidine incorporation into spleen cells from phenylhydrazine treated mice.
Spleens taken from B6C3F1 mice on the third day following the second of 2 daily injections of phenylhydrazine HCl (60 mg/kg) contain approximately 10 times the number of cells (i.e., 10(9) cells) found in spleens from normal mice. More than 90% of these spleen cells are recognizable 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 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 50-100-fold. One spleen is sufficient for 1000 triplicate Ep determinations, and the use of this microassay procedure requires only very small (1-60) microliters) 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.
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D004921:Erythropoietin / Q000032:analysis* / Q000276:immunology / Q000502:physiology; D005312:Fetal Blood / Q000502:physiology; D006801:Humans; D007106:Immune Sera; D007700:Kinetics; D051379:Mice; D008815:Mice, Inbred Strains; D010659:Phenylhydrazines / Q000008:administration & dosage*; D011817:Rabbits; D013154:Spleen / Q000166:cytology / Q000378:metabolism*; D013936:Thymidine / Q000378:metabolism
D007106:Immune Sera; D010659:Phenylhydrazines; C030299:phenylhydrazine; D004921:Erythropoietin; D013936:Thymidine