Vitronectin coating - how long last in culture?

[Seb Stephens]

I would like to coat some glass dishes with vitronectin in order to help cells
spread and image the adhesion structures.

I understand there are several factors that will affect adherance such as
density of vitronectin used per cm2 etc.

However, how long does this layer last once you have plated your cells out on
them?

Hours? Days? Weeks?

Once a cell has moved over a part of vitronectin coating, is that vitronectin
coat "used"?

My question comes from a basis that using FCS (in which I beleive vitronectin
is one of the most concentrated adhesive proteins) to coat my dishes only
seems to help cells spread when it has been newly coated, and not once the
dish has been in use for 3 days (ie, observation is that precursor cells in 3 day
samples eventually differentiate but hardly spread. If you take these
precursors and plate them on new dishes..bingo!!!!!!!!!).

sEB

[Carol Heckman]

sEB-
When you say "in use", do you mean the dish has had cells on it or has just been sitting around with buffer on it?  The layer of proteins deposited from FCS shouldn't change in composition at all after the first hour or two.  On the other hand, if the cells are there, they may add a new set of proteins or subtract proteins from the substrate, as they secrete proteoglycans and proteases.
carol
________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Waldo Schmidt [[hidden email]]
Sent: Friday, September 18, 2009 4:10 PM
To: [hidden email]
Subject: Vitronectin coating - how long last in culture?

I would like to coat some glass dishes with vitronectin in order to help cells
spread and image the adhesion structures.

I understand there are several factors that will affect adherance such as
density of vitronectin used per cm2 etc.

However, how long does this layer last once you have plated your cells out on
them?

Hours? Days? Weeks?

Once a cell has moved over a part of vitronectin coating, is that vitronectin
coat "used"?

My question comes from a basis that using FCS (in which I beleive vitronectin
is one of the most concentrated adhesive proteins) to coat my dishes only
seems to help cells spread when it has been newly coated, and not once the
dish has been in use for 3 days (ie, observation is that precursor cells in 3 day
samples eventually differentiate but hardly spread. If you take these
precursors and plate them on new dishes..bingo!!!!!!!!!).

sEB

[Seb Stephens]

Hi Carol,
 
By "in use", I do mean cells are on it.
 
Thanks for the info. I think youre right about the substrate changing once cells are on it. Id just like for it not to too quickly!
 
Seb

 


 

> Date: Sun, 20 Sep 2009 14:59:09 -0400
> From: [hidden email]
> Subject: Re: Vitronectin coating - how long last in culture?
> To: [hidden email]
>
> sEB-
> When you say "in use", do you mean the dish has had cells on it or has just been sitting around with buffer on it? The layer of proteins deposited from FCS shouldn't change in composition at all after the first hour or two. On the other hand, if the cells are there, they may add a new set of proteins or subtract proteins from the substrate, as they secrete proteoglycans and proteases.
> carol
> ________________________________________
> From: Confocal Microscopy List [[hidden email]] On Behalf Of Waldo Schmidt [[hidden email]]
> Sent: Friday, September 18, 2009 4:10 PM
> To: [hidden email]
> Subject: Vitronectin coating - how long last in culture?
>
> I would like to coat some glass dishes with vitronectin in order to help cells
> spread and image the adhesion structures.
>
> I understand there are several factors that will affect adherance such as
> density of vitronectin used per cm2 etc.
>
> However, how long does this layer last once you have plated your cells out on
> them?
>
> Hours? Days? Weeks?
>
> Once a cell has moved over a part of vitronectin coating, is that vitronectin
> coat "used"?
>
> My question comes from a basis that using FCS (in which I beleive vitronectin
> is one of the most concentrated adhesive proteins) to coat my dishes only
> seems to help cells spread when it has been newly coated, and not once the
> dish has been in use for 3 days (ie, observation is that precursor cells in 3 day
> samples eventually differentiate but hardly spread. If you take these
> precursors and plate them on new dishes..bingo!!!!!!!!!).
>
> sEB

---

Vous cherchez l'intégrale des clips de Michael Jackson ? Bing ! Trouvez !

[Carol Heckman]

Seb-
I doubt if there is anything you can do, short of killing them (and that introduces another set of problems), to keep the cells from changing their extracellular mileau.  It is one way they communicate with one another.
Carol
________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Seb Stephens [[hidden email]]
Sent: Sunday, September 20, 2009 3:26 PM
To: [hidden email]
Subject: Re: Vitronectin coating - how long last in culture?

Hi Carol,

By "in use", I do mean cells are on it.

Thanks for the info. I think youre right about the substrate changing once cells are on it. Id just like for it not to too quickly!

Seb

> Date: Sun, 20 Sep 2009 14:59:09 -0400
> From: [hidden email]
> Subject: Re: Vitronectin coating - how long last in culture?
> To: [hidden email]
>
> sEB-
> When you say "in use", do you mean the dish has had cells on it or has just been sitting around with buffer on it? The layer of proteins deposited from FCS shouldn't change in composition at all after the first hour or two. On the other hand, if the cells are there, they may add a new set of proteins or subtract proteins from the substrate, as they secrete proteoglycans and proteases.
> carol
> ________________________________________
> From: Confocal Microscopy List [[hidden email]] On Behalf Of Waldo Schmidt [[hidden email]]
> Sent: Friday, September 18, 2009 4:10 PM
> To: [hidden email]
> Subject: Vitronectin coating - how long last in culture?
>
> I would like to coat some glass dishes with vitronectin in order to help cells
> spread and image the adhesion structures.
>
> I understand there are several factors that will affect adherance such as
> density of vitronectin used per cm2 etc.
>
> However, how long does this layer last once you have plated your cells out on
> them?
>
> Hours? Days? Weeks?
>
> Once a cell has moved over a part of vitronectin coating, is that vitronectin
> coat "used"?
>
> My question comes from a basis that using FCS (in which I beleive vitronectin
> is one of the most concentrated adhesive proteins) to coat my dishes only
> seems to help cells spread when it has been newly coated, and not once the
> dish has been in use for 3 days (ie, observation is that precursor cells in 3 day
> samples eventually differentiate but hardly spread. If you take these
> precursors and plate them on new dishes..bingo!!!!!!!!!).
>
> sEB

________________________________
Vous cherchez l'intégrale des clips de Michael Jackson ? Bing ! Trouvez !<http://www.bing.com/videos/search?q=Michael+Jackson&FORM=MVDE6>

[Sylvie Le Guyader-2]

Hi Waldo

Indeed serum is full of Vitronectin so there is no need to coat your dish
with it if you have serum in your medium. I am quite sure that your dish is
still fully coated with vitronectin after 3 days with serum.

My best guess to explain the behaviour of your precursor cells is that they
express different integrins at their surface after 3 days in culture and
cannot attach to vitronectin anymore. One possibility is that they would
rather get something else that vitronectin but first use some integrins that
allow them to attach to vitronectin since that's all they can find to first
attach. Then they lay their own matrix within a few hours so you cannot have
any control of the matrix over days.

Coating the dish with different matrix proteins will modify the protein
composition of the focal adhesions at the cell/glass interface. The best set
up depends of course on what you want to do. Have you tried replating in low
(0.1-0.5%) or no serum to see if they attach? It is often assumed that you
need serum or to coat the dish for cells to attach at all but for many cell
lines this is not the case. You could replate your cells a few hours before
your experiment in a medium containing very low or no serum and see if they
adhere enough for you to image them (I assume you want to correlate adhesion
and differentiation but that might be totally wrong). Alternatively you
could seed them very sparse so that you do not need to replate them before
imaging.

Med vänlig hälsning / Best regards
 
Sylvie
 
@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader
Dept of Biosciences and Nutrition
Karolinska Institutet
Novum
14157 Huddinge
Sweden
[phone]
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Waldo
> Schmidt
> Sent: 18 September 2009 22:11
> To: [hidden email]
> Subject: Vitronectin coating - how long last in culture?
>
> I would like to coat some glass dishes with vitronectin in order to help
cells
> spread and image the adhesion structures.
>
> I understand there are several factors that will affect adherance such as
> density of vitronectin used per cm2 etc.
>
> However, how long does this layer last once you have plated your cells out
on
> them?
>
> Hours? Days? Weeks?
>
> Once a cell has moved over a part of vitronectin coating, is that
vitronectin
> coat "used"?
>
> My question comes from a basis that using FCS (in which I beleive
vitronectin
> is one of the most concentrated adhesive proteins) to coat my dishes only
> seems to help cells spread when it has been newly coated, and not once the
> dish has been in use for 3 days (ie, observation is that precursor cells
in 3 day
> samples eventually differentiate but hardly spread. If you take these
> precursors and plate them on new dishes..bingo!!!!!!!!!).
>
> sEB