In vitro assays are detrimental to determine cellular function. During my PhD studies I used many in vitro readouts for cellular function and metabolism of macrophages to find novel targets to treat chronic inflammatory disorders like atherosclerosis. The assays I used mostly involved 96-well based assays to quickly analyze changes in cell response upon several activators and inhibitors.
Before we acquired the Cytation 5 from BioSPX, we mostly quantified our readouts based on absorbance or by flow cytometry, but we could not quickly scan for visual changes or cell numbers in this 96-well format. As such, we did not normalize cytokine or NO secretion for cell counts and could only normalize metabolic flux analyses for protein or DNA abundance in the well.
Measurements from absorbance to fluorescent pictures
In 2020, after obtaining a so-called equipment grant, our lab bought the cytation 5 and I’ve extensively used it since. In the first place it was the perfect tool to normalize our extracellular flux analyses by injecting cell-permeable Hoechst in the last step of the assay and imaging and counting with the cytation. In our paper (Verberk, de Goede et al. 2022, cell reports methods), we validated that this method decreased standard deviations. Additionally, the Cytation also helped me to normalize ELISA data with brightfield images from valuable patient monocyte-derived macrophages and to visualize with fluorescent pictures Bodipy-based lipid staining before measuring with flow cytometry.
All in all, for my research projects, the Cytation 5 came out very handy and useful in several ways. As it is able to measure from absorbance to fluorescent pictures, it is broadly applicable to many in vitro assays.
PhD Sanne Verberk
Department of Molecular Cell Biology and Immunology
Amsterdam UMC, VUmc