hi everybody,
i have some troubles with chemokines and cell proliferation. Different papers evidence that CXCL8 (but also CXCL12, CCL2 and other chemokines) are able to promote cancer cell proliferation. Based on these statements i tried to assess if CXCL8 would increase TPC-1 and 8505C thyroid cancer cell proliferation in vitro. Firstly i seeded my cells in a 96 well flat at a density of 4000 cells x well. After adhesion i treated cells with increasing concentrations of CXCL8 (10, 50 and 100ng) for 24 48 and 72h . At the end of each time of treatment i stained cells with cristal violet 0.5% fixed with METOH. I read the OD and calculated my results basing on control untreated. However my results were not reproducible. I tried also to assess the proliferation by CFSE at FACS but i did not found any increase of cell proliferation after treatment with CXCL8. I do not understand what's wrong, i have probably choose not adequate proliferation assays? could anyone suggest me some alternatives?
Thankyou
Francesca
Hi Francesca,
Alamar blue, trypan blue, BrdU staining should work. Also, crystal violet fixed with methanol should be fine, but maybe more troublesome. To me it seems that the CXCL8 quantity you use is very low, hence maybe ineffective.
Another point to add just in case you are not following this methodology, when you wanna start your measurements from 0 hour timepoint, at that timemake sure you remove your serum-filled media and substitute with serum-free media, otherwise you will have other factors too inducing proliferation and you won't be able to conclude whether CXCL8 or serum is inducing proliferation.
Thankyou Aggelos,
I Will try removing serum firstly! When you say Trypan blu do you mean counting with burker chamber?
Yes, e.g .see:
Maxwell PJ, Neisen J, Messenger J, Waugh DJ. Oncotarget. 2014 Jul 15;5(13):4895-908.
or
Broxmeyer HE, Cooper S, Hangoc G, Kim CH. Stem Cells Dev. 2005 Apr;14(2):199-203.
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