Hi,
I was wondering if you could suggest me any assay which deal with (endothelial) cell proliferation in vivo apart from BrdU labelling. I have been told that I may find other routine assays regarding that matter.
There are a number of assays that can be suggested. I personally like tritiated thymidine and have published a couple of papers using it. Some people use MTT. It is more of a marker of cell metabolism but has been substituted for proliferation as well. Most large scientific retailers offer some type of assay. Invitrogen/Life Technologies/Fisher has a cell dye called cell trace that can be used on a flow cytometer. Pick your pleasure. I believe the MTT is the cheapest route.
Thank you Torry. But I am also looking for in vivo tracing for (endothelial )cell proliferation assays. If I am right, MTT assay is an approach for cell proliferation in vitro
Cy - quant assay should work, unlike MTT it measures absolute DNA. Ideal for variety of adherent cells. You will need a fluorescence plate reader. Goodluck
Hi,
We use a Click-iT EdU assay. Similar conceptually to BrdU, but much simpler. Though we only used it as an in vitro assay, I have read about it being used within an in vivo setting.
See page 15/16 on the link...
Good luck!
http://www.unicaen.fr/services/cmabio/pdf/proliferation-measurement.pdf
We use Ki-67 as a marker of proliferating cells. You can use confocal immunofluorescence to show co-localization of endothelial cells and proliferating cells.
You can analyze cell proliferation of HUVECs cells using CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS).
You can also analyze cell proliferation using CellTrace™ CFSE Cell Proliferation Kit | Life Technologies . Both assay works very well for HUVECs.
if in vivo tritiated thymidine would be first choice then BrDu. Ki67 fine if don't want to label. Native mitoses can also work or can arrest cells going into mitosis with vincristine. Must consider compartment size as labelling indices can be misleading if both sides of the ratio change. See my review in Cell Proliferation Cell Prolif. 2007 Apr;40(2):231-40
You can use Ki67 if you're just counting the cells. MTT as others have suggested is a metabolism/cell viability assay and not really a proliferation assay. Some reviewers may find it unacceptable for a proliferation assay (one of my advisors, for example), some may be ok with it, but I wouldn't use it for proliferation.
Brdu is a convenient assay for checking cell proliferation for endothelial cells. Except brdu, you can go for
1. MTT assay
2. SRB assay
3. Thymidine incorporation assay
4. Cell counting assay by tryphan blue or by kits etc.
Hi
I just want to thank you all for your valuable helps.
Try cell survival assay from Roche. This assay measures the mitochondrial activity and one could directly co-relate that with cell proliferation.
Also, why not BrdU. It works pretty well with endos. Did you find it difficult using BrdU?
Hi Amjad, I am planning to evaluate EC proliferation in vivo and I am looking for other approaches to do so apart from BrdU. Peoples above have been very helpful. If you could suggest me also any other methods I would be very thankful.
I guess Endoglein is a marker of proliferating endothelial cells, you may even perform duel IHC for endoglein and ki67. Hope that works out well for you. Thanks
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