A rapid spectrophotometric method to identify inhibitors of human erythropoiesis

Erythropoiesis is a complex physiological process by which erythroid progenitors proliferate and differentiate into nonnucleated red blood cells. Several methods can be used to monitor in vitro the differentiation of erythroid precursors, and hence the toxic effects of drugs, chemicals, or pollutants. One of the most commonly available assay of erythropoiesis is the microscopic observation of differentiated cells after benzidine staining, which forms a blue complex with hemoglobin. However, this method is laborious and does not provide accurate results since it heavily relies on the reader's interpretation. Moreover, benzidine is a carcinogen and a highly reactive molecule which forces the reader to microscopically count differentiated and non-differentiated cells within a short time frame (5 min). Here we have developed a simple, inexpensive, in-vitro spectrophotometric assay to measure erythroid differentiation using K562 cell line as a model. Materials needed included 96-well round-bottomed microplates and a microplate reader. Remarkably, carcinogenic benzidine was replaced by its isomeric tetramethyl derivative, the 3,3', 5,5'- tetramethylbenzidine (TMB), which presents several advantages: it is cheap, not mutagenic and a ready-to-use chromogenic substrate. A small volume (50 μl) of TMB added to the samples forms a blue complex in 15 min, and the reaction can be easily stopped and stabilized by the addition of H₂SO₄. The yellow precipitate is then solubilized, and the absorbance is measured at 450 nm. In addition, the suitability of the assay to determine the effects of compounds on erythroid differentiation was further tested with known inhibitors (artemisinin derivatives) of K562 differentiation. Overall, the reported methodology permits to measure in an accurate and reproducible manner the K562 differentiation and can be used for medium throughput screenings (MTS) of compounds or environmental toxics with potential erythro-toxicity and ability to inhibit erythroid differentiation.