I will have to do a lot of tube formation assay in the near future and hoping to automate/semi-automate this for ease of imaging and quantification . I do not want to fix my cells as I am hoping to see the effect of my test compounds on angiogenesis over a period of time. For this purpose, I am looking for a stain that is not interfering with FCS (or any other components of media), which is stable for at least 24 hrs and is transferred from the mother to the daughter cell. Any input will be much appreciated.
You best try 2uM calcein AM. It stains live cells, in full medium, and within 10 minutes. To my knowledge, it does not interfere with cell function, and is frequently used in these types of assays (Check the angiogenesis assays from BD biosciences for example). You might need to refresh the medium when you expect much proliferation (but this will obscure the effects on tube formation!).
Also, be aware that after fluorescent imaging, the free radicals that arise will have a negative effect on cell survival.
Best,
Judy van Beijnum
VUmc, Amsterdam, Netherlands
www.angiogenesis.nl
I use Calcien AM we make a 4mM stock store at -12oC in dark. To use dilute 1.6ul of 4mM stock in 2 ml of your media and resuspend your cell pellet in this. Incubate the cells in suspension for 10min at 37oC then spin your cells down again. You should see a green cell pellet. CalcienAM passes freely through cell membranes and becomes Calcein AM ester which then can not leave the cell. I get good images in 24 hours.
Caroline, Do you buy your calcein from Invitrogen? Also, what is the cell density for the staining that you have described above?
Hi Ayse
The labeling should be fine for a confluent T75 flask of cells as the dye is in excess. This dye will pentrate all cells either in monlayer or suspension
Hope this answers your question
Caroline
Carboxyfluorescein succinimidyl ester (CFSE) is an other possibility. I used to stain my cells with it before putting them in the gel for culture. I could follow them building structures at least for a week.
I have used both direct staining as described by others here but have also successfully used CellLight products from Invitrogen, to pre-label the cells. CellLight is a baculoviral system with very simple application protocol. I used an actin probe as label but there are a variety. I would also echo the comments of others here that endothelial cells are quite sensitive to excessive or regular fluorescent exposure and you might wish to perform a calibration comparison of your assay with and without fluorescence exposure prior to any crucial experiments.
We have used the Vybrant DiO, DiI and DiD dyes from Life Technologies to pre-label endothelial cells from different sources/origins to examine their ability to collaborate subsequently in tubule formation. These did not appear to impair tubule formation, whereas the lipophilic dyes such as PKH26 or PKH67 did appear to interfere to some extent with tubule formation.
Hi!
You could use CellTracker dye, Dil, (Invitrogen) for staining the live cells. It lasts as long as 100 days. This dye dose not has bad effects on any cells specially Endothelial cells but you must be care of using the optimum dose not more, because high amount of dye reduce the cell proliferation.
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