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Need advice on Adhesion Assay


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#1 Pronana

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Posted 06 March 2009 - 09:17 PM

Hello experts,

I am planning to do a cell adhesion assay to study some plant extract's ability to inhibit cancer cell adherence on fibronectin in a 96 well plate. I have came across various protocols and was hoping someone might iron out some confusion i have.

These protocols require me to coat the plates with fibronectin followed by blocking any remaining binding sites on the plate with BSA .

Can someone tell me what is the blocking agent for and what site specifically am I blocking if i block the binding sites then how would the cells adhere? Is it possible for me to use precoated fibronectin plates instead of coating them on my own?If i use precoated fibronectin plates, do I still need to block the plates prior to adding cells for this assay?

Thank you in advance for you kind attention.

#2 WOW

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Posted 15 March 2009 - 08:10 PM

BSA could cover binding sites both on FN layer and non-fibronectin place in your dish (since coating by hand can't cover all places in your dish). Since your cells have a extra high affinity, if there is, it would be advantageous to competite with BSA to adhere on FN. And BSA and your cell was assumed to poccess similar ability to adhere on your dish (non-FN coating sites), therefore your cell should not be adhere on non-FN coating places in your dish (turned out to be false positive, if BSA not used). NO BSA were needd in commercial FN-coated dish, I guess (You just paid money for this). May it help you and good luck.
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#3 Pronana

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Posted 23 June 2009 - 03:39 AM

BSA could cover binding sites both on FN layer and non-fibronectin place in your dish (since coating by hand can't cover all places in your dish). Since your cells have a extra high affinity, if there is, it would be advantageous to competite with BSA to adhere on FN. And BSA and your cell was assumed to poccess similar ability to adhere on your dish (non-FN coating sites), therefore your cell should not be adhere on non-FN coating places in your dish (turned out to be false positive, if BSA not used). NO BSA were needd in commercial FN-coated dish, I guess (You just paid money for this). May it help you and good luck.



Dear sir,

Are you saying that the BSA coating is to block the binding of the cells on non fibronectin sites? If BSA has affinity to adhere to FN, wouldn't coating the wells with BSA block the adherence of the cells to the FN completely?

I have purchased a set of commercial coated 96 well fibronectin plates with one row of the wells coated with BSA while the rest is coated with FN (RnDSystems). What do the adherence of cells to BSA coated wells supposed to indicate?


I am at wits end and i would be grateful if you could shed some light. :)

#4 eldon

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Posted 01 July 2009 - 10:59 AM

I am planning to do a cell adhesion assay to study some plant extract's ability to inhibit cancer cell adherence on fibronectin


If BSA has affinity to adhere to FN, wouldn't coating the wells with BSA block the adherence of the cells to the FN completely?


fibronectin is secreted correct? many other secreted ecm glycoproteins that are laid down can be used by the cell or neighbouring cells in a manner that might modulate adhesion.

coating the plate with FN will not be 100% effective, and so uncoated spots will be available for the cell(s) to adhere to OR for other secreted adhesion proteins to adhere...these 2 scenarios are controlled for to some extent by using excess BSA to block these sites...if left unblocked, your data might be difficult to interpret, repeat or just be inaccurate...you want to be sure your extract of interest inhibits cell-FN adhesion and that any affect on adhesion you measure is not simply due to the heterogenous lack of FN on the surface of the dish.

in a nutshell:

1. u r measuring cell-FN adhesion in presence of extract
2. experiment -> add cells to FN coated dishes (positive ctl), add cells to FN coated dishes plus boiled extract (negative ctl-boil should denature protein factors), add cells to FN coated dishes plus extract (test)
3. blocking plates with BSA increases the probability that the measured effect is due the extracts ability to alter cell-FN adherence and not cell-dish adherence or cell-non FN adhesion

#5 Pronana

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Posted 07 July 2009 - 04:49 AM

Dear eldon,

Thank you for your feedback. I get it now. However, is it a must for me to still block the coated wells with BSA if i have bought precoated plates?

#6 Prince

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Posted 23 September 2009 - 03:42 PM

Hello experts,

I am planning to do a cell adhesion assay to study some plant extract's ability to inhibit cancer cell adherence on fibronectin in a 96 well plate. I have came across various protocols and was hoping someone might iron out some confusion i have.

These protocols require me to coat the plates with fibronectin followed by blocking any remaining binding sites on the plate with BSA .

Can someone tell me what is the blocking agent for and what site specifically am I blocking if i block the binding sites then how would the cells adhere? Is it possible for me to use precoated fibronectin plates instead of coating them on my own?If i use precoated fibronectin plates, do I still need to block the plates prior to adding cells for this assay?

Thank you in advance for you kind attention.


Hi all:

I am new to this forum. Why do we starve the cells from serum before the cell adhesion assay?Can any one explain!

Thanks,
Prince

Edited by Prince, 23 September 2009 - 03:45 PM.





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