Université de Fada N'gourma, Institut Supérieur du Développement Durable, BP 54 Fada N'gourma, Burkina Faso; Centro Interuniversitario di Ricerca sulla Malaria-Italian Malaria Network (CIRM-IMN), Milano, Italy.
Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Via Pascal, 36, 20133 Milano, Italy; Centro Interuniversitario di Ricerca sulla Malaria-Italian Malaria Network (CIRM-IMN), Milano, Italy.
J Pharmacol Toxicol Methods. 2022 Jan-Feb;113:107134. doi: 10.1016/j.vascn.2021.107134. Epub 2021 Nov 17.
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 HSO. 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.
红细胞生成是一个复杂的生理过程，在此过程中，红系祖细胞增殖并分化为无核红细胞。有几种方法可用于监测体外红细胞前体的分化，从而监测药物、化学物质或污染物的毒性作用。最常用的红细胞生成检测方法之一是使用联苯胺染色后对分化细胞进行显微镜观察，联苯胺与血红蛋白形成蓝色复合物。然而，这种方法既繁琐，结果又不准确，因为它严重依赖于观察者的解释。此外，联苯胺是一种致癌物质和高反应性分子，这迫使观察者在很短的时间内（5 分钟）对分化细胞和未分化细胞进行显微镜计数。在这里，我们开发了一种简单、廉价的体外分光光度法，使用 K562 细胞系作为模型来测量红细胞分化。所需的材料包括 96 孔平底微孔板和微孔板读数器。值得注意的是，致癌的联苯胺被其异构体四甲基衍生物 3,3',5,5'-四甲基联苯胺（TMB）取代，TMB 具有以下优点：便宜、非诱变、现成的显色底物。向样品中加入少量（50 μl）TMB，15 分钟后形成蓝色复合物，通过加入 HSO 可以很容易地停止和稳定该反应。然后将黄色沉淀物溶解，并在 450nm 处测量吸光度。此外，该测定法还可用于测试已知的 K562 分化抑制剂（青蒿素衍生物）对红细胞分化的影响，进一步证明了该测定法的适用性。总的来说，所报道的方法允许以准确和可重复的方式测量 K562 分化，并且可以用于化合物或具有潜在红细胞毒性和抑制红细胞分化能力的环境毒物的中高通量筛选（MTS）。
