Counting nuclei released from microcarrier-based cultures using pro-fluorescent nucleic acid stains and volumetric flow cytometry

Abstract

Counting nuclei released from intact cells is a convenient and reliable approach to assess cell density during microcarrier-based culture of adherent cells. However, commonly used methods for counting nuclei, such as crystal violet staining and quantification with a hemocytometer/ automated imaging system or a Coulter counter, are imprecise, labori­ous and, limited in throughput. Here, we describe the use of high-affini­ty pro-fluorescent nucleic acid stains and volumetric flow cytometry for automated counting of nuclei released from cells attached to microcar­riers with improved precision and high sample throughput. This simple procedure facilitates rapid and precise assessment of cell attachment, survival, and proliferation on microcarriers, and can provide informa­tion about the cell cycle, all without the need for cell detachment. Con­sequently, various microcarrier-based applications, from small-scale multi-factor experiments to large-scale functional genetic screens and clinical/industrial cultures, could be enhanced by this approach.