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SeedEZ 3D cell culture application notes - microplate reads. The users of 3D cell cultures often wonder how to read and how to interpret readout results obtained with microplate readers. Understanding a microplate optical system may be complex. Lena Biosciences provides simple explanations that any cell culture user interested in using a plate reader, whether with 2D or 3D cell cultures, understands.
Rest assured, reading from a three-dimensional (3D) cell culture is not too different than reading it from a cell monolayer. 3D cell cultures are compliant with most plate readouts and detection methods when cell lysates, extracted proteins, metabolic products or other solutions are transferred into another plate and read. We refer to these reads as the post-transfer reads, as they are obtained following the transfer of cell lysates, excreted molecules etc., into a new multi-well plate in which the cells are not present. For the most consistent results, you may want to shake the microplate with a 3D cell culture prior to solution transfer. Provided that a reasonable intra-3D-cell-culture mass transport takes place, most post-transfer reads allow for direct comparison between 2D and 3D cell culture models.
Today, frequently used cytotoxicity, viability, proliferation and chemo-sensitivity assays with 3D cell cultures are Alamar Blue, CellTiter-Glo, Neutral Red and Sulforhodamine B (SRB) assay. An advantage of Alamar Blue assay is that it is not terminal. If you are interested in assessing delayed cell death for the period of days following toxic compound withdrawal, non-cytotoxic and non-terminal cell metabolic activity assays may be appropriate.
For the in situ reads, with 3D cell culture present in the plate wells, an absorbance read may be less appropriate than a fluorescence read or a luminescence read. Next, the plate reader illumination and detection optics arrangement may favor one cell culture model vs. the other. For example, the top epifluorescence reads often provide better signal-to-noise ratio for solution-based assays. The bottom reads may provide better results when working with an adherent layer of cells. This complicates direct comparison between in situ reads with cells in 2D and 3D cell cultures. Please refer to this application note to learn more. It provides step-by-step instructions, comparisons and results for different optical arrangements and teaches what should be taken into consideration when comparing 2D and 3D in situ cell culture reads.
As always, setup suitable controls; avoid using outer wells in 96-well plates and higher - fill them with water or media to prevent evaporative losses; and always compare results obtained only with the identical microplates and the identical readout conditions.