I'm currently trying to perform an in vitro alamar blue assay using neonatal fibroblast cells. Unfortunately I'm encountering trouble visualizing the stained adherent cells with the microscope, and inaccurate OD readings, most likely due to the thickness of the gel treatment. I tried seeding the cells first, then adding the hydrogel treatment on top. Does anyone have any insight on how I might test the effects of the hydrogel using this method?
none of my hydrogel plated samples were reduced probably due to inability for the dye to be taken up due to the hydrogel obstruction.
Please advise
Thanks for your time
Dear friend Salma Younes
It sounds like you are encountering some challenges in performing an in-vitro alamar blue assay with neonatal fibroblast cells following hydrogel treatment. There are a few potential issues that could be causing your difficulties in visualizing the stained cells and obtaining accurate OD readings.
Firstly, it is possible that the hydrogel treatment is interfering with the alamar blue dye uptake by the cells. If the hydrogel is too thick, it may prevent the dye from diffusing to the cells or interfere with the reduction reaction that produces the color change. In this case, you could try adjusting the thickness of the hydrogel to improve dye diffusion, or considering a different dye that may be better suited to your specific hydrogel system.
Another potential issue could be related to the timing of your assay. It is important to ensure that the hydrogel treatment has fully equilibrated, and any potential cytotoxic effects have dissipated before performing the assay. Depending on the specific hydrogel system you are using, this may require a longer incubation period before adding the alamar blue dye.
In terms of visualizing the stained cells, it is possible that the thickness of the hydrogel is making it difficult to image the cells with the microscope. In this case, you could consider using confocal microscopy or other imaging techniques that may be better suited to imaging through thick samples.
Overall, it may be helpful to optimize your assay conditions, including hydrogel thickness, incubation time, and imaging techniques, in order to improve the accuracy and reproducibility of your results. Additionally, you may want to consider consulting with colleagues or seeking out relevant literature to see if others have encountered similar challenges with alamar blue assays following hydrogel treatment.
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