I want to see the expression of endothelial secreted molecule in MDA-MB-231 Breast cancer cells. I am curious about the expression whether i should co-culture with endothelial cells
Please give some suggestion regarding this .
Thank you
If the molecule you want to check is a protein, you can easily assess it's presence in MDA cells by flowcytometry. You would need just 3 antibodies: one against the protein of interest and the other two against surface markers expressed on the two cell types (MDA and endothelial) in order to discriminate them. Obviously, the 3 antidobies must be labelled with different fluorochomes.
Keep in mind that in any case you'd check the presence/absence of the protein. If it has been demonstrated that this protein is secreted by endothelial cells and can be uptaken by MDA cells everything is fine. Otherwise, you should demonstrate that endothelial cell produce the protein, thus you should at least measure protein concentration in the medium with and without endothelial cells using Elisa or western blotting (other experiments inhibiting the protein synthesis may be required). Also, you should demonstrated that the protein is actually uptaken by receptor blocking assays. Indeed, the increasing could be due also by an up-regulation of transcription/translation in MDA cells induced by other soluble factors as well as cell-cell contact, and not by the direct uptake. Good luck!
Costanza Maria Cristiani Dear madam, Thank you for the valuable answer. you specified some valuable points.
Thank you
culture endothelial cells in 6 well plate, make 3D spheroids of mda mb 231. co culture it.
isolate both separate.
first wash to take mda mb 231 using pbs buffer. later apply trypsin for endothelial cel.
use crl4003 as nor cell line for endothelial or
ishikawa endometrial cancer cell line.
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