Simply red: A novel spectrophotometric erythroid proliferation assay as a tool for erythropoiesis and erythrotoxicity studies

Abstract

Most mammalian cell proliferation assays rely on manual or automated cell counting or the assessment of metabolic activity in colorimetric assays, with the former being either labor and time intensive or expensive and the latter being multistep procedures requiring the addition of several reagents. The proliferation of erythroid cells from hematopoietic stem cells and their differentiation into mature red blood cells is characterized by the accumulation of large amounts of hemoglobin. Hemoglobin concentrations are easily quantifiable using spectrophotometric methods due to the specific absorbance peak of the molecule’s heme moiety between 400 and 420 nm. Erythroid proliferation can therefore be readily assessed using spectrophotometric measurement in this range. We have used this feature of erythroid cells to develop a simple erythroid proliferation assay that is minimally labor/time- and reagent-intensive and could easily be automated for use in high-throughput screening. Such an assay can be a valuable tool for investigations into hematological disorders where erythropoiesis is dysregulated, i.e., either inhibited or enhanced, into the development of anemia as a side-effect of primary diseases such as parasitic infections and into cyto-(particularly erythro-) toxicity of chemical agents or drugs.