Most quantitative assays for cell proliferation are based on end-point measurements (EdU or BrdU incorporation, Ki67 staining, etc). However, several alternatives have been developed to measure cell proliferation using non-toxic fluorescent cell dyes (see this poster from Nikon, http://www.nikon.com/products/instruments/lineup/bioscience/integrated/ct/pdf/Estimation_of_Stem_Cell_Numbers.pdf), holographic imaging (http://www.phiab.se/applications/live-cell-imaging), or the FUCCI system (http://www.lifetechnologies.com/us/en/home/life-science/cell-analysis/cell-viability-and-regulation/cell-cycle/live-cell-imaging-of-cell-cycle-and-division.html). All these methods require some level of specialized microscopy, so translucency and intrinsic fluorescent properties of your polymers could be limiting.

If the latter case proves true, then you could probably adopt a high-throughput assay by lysis of your cells at different times, and quantifying total DNA by Cyquant on a plate reader. Would revert to end-point assays, but there may be no better option if your system is not compatible with microscopy.

Hi, Christopfer, if you have a good fluoresent microscope, you can try couple of fluoresent marker relate with cell proliferation. Fluoresent dyes, as Daniel mention, is also a good idea. But in some case dyes may have an effect on cell prolifaretion, if you incubate it for a long time.

In ideal case you can create a tarcking system whcih can take images automatically once per 20-30 minutes, for example. If you have histone:GFP marler. you can easy quantifies cell divsion ratio, as well as cell cycle duration etc.

But you need to have a precise automatical tracking system..

Good luck!

Cell titer blue or alamar blue can be used for this. It's a non-toxic dye and travels across the cell membrane so no fixing or lysing is necessary, but you have to be sure you thoroughly rinse afterwards (several media changes should be fine) and use the same protocol, gain, etc.

For these type of experiments we usually use CellTiter Proliferation Assay. https://www.promega.de/products/cell-health-and-metabolism/cell-viability-assays/celltiter-96-aqueous-one-solution-cell-proliferation-assay-_mts_/?origUrl=http%3a%2f%2fwww.promega.com%3a81%2fproducts%2fcell-health-and-metabolism%2fcell-viability-assays%2fcelltiter-96-aqueous-one-solution-cell-proliferation-assay-_mts_%2f It is an easy to do colorimetric method. The Assays are performed by adding a small amount of the CellTiter directly to culture wells, incubating for 1–4 hours and then recording mitochondrial activity by absorbance at 490nm with a 96-well plate reader. The quantity of formazan product as measured by the amount of 490nm absorbance is directly proportional to the number of living cells in culture.

Another method, which we use in case the CellTiter Reagent is crossreacting with polymers or scaffolds is neutral red. https://www.researchgate.net/publication/5251539_Neutral_red_uptake_assay_for_the_estimation_of_cell_viabilitycytotoxicity

The neutral red uptake assay is based on the ability of viable cells to incorporate and bind neutral red in their lysosomes. It is also easy to do: Cells are incubated for 4 h with a medium containing 0.05% neutral red. The cells were subsequently washed with PBS. Afterwards, the cells are checked under the microscope and then the dye is extracted with (100 μl) desorb solution in each well and the absorbance was read using the 96 well-plate reader (Anthos, Microsystem, Germany) at 540 nm. Desorb solution: 50% Ethanol, 49% H2O, 1% Acid Acetic. As it is suggested by Repetto et al. 2008. I recommend to perform a standard curve for both assays.

Article Repetto G, del Peso A, Zurita JL.. Neutral red uptake assay ...

To work without dye or other chemical, we use the x-celligence system that report in real time the proliferation of adherent cells. It works with plates lined with an electrode measuring electrical impedance that estimates the surface of the wells occupied by cells.

http://www.aceabio.com/product.aspx

You can then have proliferation curves for multiple wells in real time without any chemicals.

Although you have to seed the cells directly on the plate (not on a coverslip), you might want to check if it is possible to place a matrix between the cells and the bottom of the well. Plates with "windows" are available: a part of the bottom's surface is devoid of electrode, this way you can monitor either cell morphology or staining of the cells (we do immunofluorescence).