Abstract
Cell quantification assays are essential components of most biological and clinical labs. However, many currently available quantification assays, including flow cytometry and commercial cell counting systems, suffer from unique drawbacks that limit their overall efficacy. In order to address the shortcomings of traditional quantification assays, we have designed a robust, low-cost, automated microscopy-based cytometer that quantifies individual cells in a multiwell plate using tools readily available in most labs. Plating and subsequent quantification of various dilution series using the automated microscopy-based cytometer demonstrates the single-cell sensitivity, near-perfect R2 accuracy, and greater than 5-log dynamic range of our system. Further, the microscopy-based cytometer is capable of obtaining absolute counts of multiple cell types in one well as part of a co-culture setup. To demonstrate this ability, we recreated an experiment that assesses the tumoricidal properties of primed macrophages on co-cultured tumor cells as a proof-of-principle test. The results of the experiment reveal that primed macrophages display enhanced cytotoxicity toward tumor cells while simultaneously losing the ability to proliferate, an example of a dynamic interplay between two cell populations that our microscopy-based cytometer is successfully able to elucidate.
Highlights
Cell quantification assays are essential components of most biological labs, and are used for a variety of applications, including cytotoxicity, viability, and proliferative studies
Current cell quantification assays can be divided into two major classes: metabolic and cell counting
J774.A1 mouse macrophages were obtained from ATCC and maintained in Roswell Park Memorial Institute Medium (RPMI), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin
Summary
Cell quantification assays are essential components of most biological labs, and are used for a variety of applications, including cytotoxicity, viability, and proliferative studies Though these assays have improved significantly since the advent of hemocytometers, they still suffer from a number of apparent limitations. They are not ideal for certain experimental setups, as they have a limited dynamic range and are prone to confounding interference in the presence of certain chemicals (Chakrabarti et al, 2000; Doak et al, 2009; Hamid et al, 2004; Ulukaya, Colakogullari & Wood, 2004; Vistica et al, 1991). Often considered the gold standard for cell counting and analysis, is an especially powerful technique for quantifying individual cells, and is one of the few modalities capable of identifying cell population counts in both a mono-culture and co-culture setup (Gedye et al, 2014; Gerashchenko, 2008; Gerashchenko & Howell, 2013)
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