I am culturing epithelial organoids (isolated from small intestine) in matrigel. I would like to study proliferation. Does anyone know which proliferation assay is the most suitable for such culture?
You could check out "A reliable tool to determine cell viability in complex 3-d culture: the acid phosphatase assay" publshed by Friedrich et al. in Journal of Biomolecular Screening in October 2007, 12 (7) :925-37. The Invitrogen website for AlgiMatrix(R) 3D culture systems recommends the use of Alamar Blue(TM) to measure cell viability. You could buy Alamar Blue(TM) or buy the active ingredient, resazurin, and look at mitochondrial oxidoreductase activity as a measure of cell viability.
I use MTS assay regularly while assessing viability in oesophageal spheroids, but I culture them in Happy Cell
We use an Optronix GELCOUNT, very useful and worth the money if can get access to one and plan to do a lot of these (and other people in your lab?). It can do varied plate formats (6-well to 96-well), on live cells also. Otherwise its a fix and stain job (eg crystal violet) followed by manual counting, much like a standard colony formation assay (aka clonogenic assay).
You could check out "A reliable tool to determine cell viability in complex 3-d culture: the acid phosphatase assay" publshed by Friedrich et al. in Journal of Biomolecular Screening in October 2007, 12 (7) :925-37. The Invitrogen website for AlgiMatrix(R) 3D culture systems recommends the use of Alamar Blue(TM) to measure cell viability. You could buy Alamar Blue(TM) or buy the active ingredient, resazurin, and look at mitochondrial oxidoreductase activity as a measure of cell viability.
CellTiter GLO from Promega, which quantifies intracellular ATP by measurement of emitted light via luciferase activity in a plate reader works very well for assessing proliferation of spheroid cultures. Using it a lot for neurosphere assays.
It sort of depends on whether you need to assess proliferation as an endpoint or as a part of another assay. It also depends on what type pf grainularity you need in the data. The suggestions here are all good, but MTS and other metabolic assays are indirect, and may or may not accurately reflect actual cell number as spheroids grow. However, if you want a gross estimate of cell number those will be fine, given that caveat, and that they are endpoint assays usually. Colony counters are also nice, but again aren't likely to give you very precise cell numbers, but can be done as non-endpoints (stick the plate in the counter and take it back to culturing). As I said though, these are both great suggestions, and pretty easy. Just to add another to your pile of choices, we like topro-3 dyes, since they can be used in live cells, are linear with ds-dna content, and so report a closer approximation of cell number/prolifereation, rather than just cell viability, etc. Another advantage is that since it is in the far red channel, it leaves open visible channels for subsequent fix and staining or fusion protein expression. The downsides are primarly two: (1) it will also stain dead cells, if you have a lot that will be a problem, and (2) there may be some background in matrigel; I have used it successfully with collagen gel cultures with minimal background.
@ Robert, you are completely right - I am rather thinking about proliferation as an endpoint. For the FACS-based methods I need to have single cell suspension and this is at the moment difficult point. After organoid digestion I get mixture of cells: single cells, doublets etc. Unfortunately my organoids are very sensitive so I cannot use e.g. trypsin. Anyway I will try to improve this single cell dissociation... Thank you for the topro-3 dye idea, I will take it into account. I would like to ask you what do you think about usage of EdU?
@ Shailendra , I am wondering whether the presence of matrigel at the bottom of the well will not interfere with the plate reading (fluorescence/ absorbance)?
well regarding the background fluorescence/absorbance, again that is a nice thing about using far red dyes- they have little of either in the UV or low viz wavelengths. Although as always, one would of course take a backgroun/zero of a sample with no cells, right? ;)
Couldn't you use the Crystal Violet Assay?
"With crystal violet this problem of the fibroblasts influencing
the outcome of the assay can be overcome by lowering the pH of
the dye. It has been shown that at pH 2.8 the epithelial specificity
of the stain increases, giving a more accurate analysis of epithelial
cell number
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