Coating Protocol

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Coating Protocol

Post  Steve on Thu Aug 07, 2008 2:20 am

Dear MCS,





My lab has recently switched from AYA to MCS MEAs. Our old coating protocol, which worked well
with AYA, does not seem to be working with our new 200/30 ITO MEAs. The neurons
tend to clump up, and do not adhere to the surface well, as if it were not
coated at all. Can you perhaps critique our current coating procedure?

Current method:


Sterilize the MEAs via autoclave


Treat them with FBS to make the surface hydrophilic,


Wash with water


Treat with Poly-Ornathine to establish a basal ECM


Aspirate and let the MEA dry before washing with water


Apply Laminin a few hours to overnight before plating
neurons on it





Cleaning: Terg-a-zyme for 3 hours at room temperature





Thank you,




Steven

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Re: Coating Protocol

Post  Steve on Thu Aug 07, 2008 2:26 am

Oh - forgot to add: the neuron types are
disassociated (not a complete slice) E17 mouse cortical or human embryonic
derived. We tend to let them sit
from 3-6 days after plating before recording.

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Re: Coating Protocol

Post  Steve on Thu Aug 07, 2008 3:48 am

And another update apparently we are using the
protocol indicated in the MEAs users manual.
The problem we see cells clumping up and dying without adhering
seems to look like the surface is too hydrophobic. Weve had the same problem with AYAs MEAs, but we got rid of
that with the FBS. Is it possible that
our new MCS MEAs just need more FBS treatments?

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Hydrophilization

Post  ThomasMCS on Mon Aug 11, 2008 7:11 pm

Dear Steve,

please apologize our delayed reply.
To me it seems, that the surface of the MEAs you are using now is hydrophobic. This is the same as with the Ayanda MEAs. There are two options to achieve a more hydrophilic surface. Either a high concentration of protein (such as FBS) or (even better) by plasma treatment. We do routinely apply plasma cleaning to the MEAs used in our lab to ensure a reproducible hydrophilization. FBS works, but with higher varaibility...

Greetings

Thomas

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Re: Coating Protocol

Post  Steve on Tue Aug 12, 2008 3:35 am

Thank you for the reply.

I was wondering what plasma chamber equipment you use? We have an oxygen plasma chamber (I do not remember the make or model), and a hand held high frequency high voltage "plasma gun" that can be used to generate ozone. The hand held device tends to blow up AYA MEAs, though.

Thanks,

Steve

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Plasma Cleaner

Post  ThomasMCS on Tue Aug 12, 2008 6:31 pm

Dear Steve,

we use a plasma cleaner from Harrick
http://www.harrickplasma.com/products_cleaners.php

I have no Idea about the plasma gun you are using.
Maybe you can try on an old MEA to see if our chips tolerate it.

Thomas

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Re: Coating Protocol

Post  Steve on Wed Feb 18, 2009 12:03 pm

Another lab in our bulding has the exact same plasma chamber. For how long do you treat it for? I'm trying out some spare (dead) AYA MEA's for 2 minutes, and am seeing cracking in the grounding electrode.

Steve

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Re: Coating Protocol

Post  ThomasMCS on Wed Feb 18, 2009 6:54 pm

We usually treat the MEAs for 1-2 minutes.
Is it possible that the Plasma Cleaner you used is not working with regular air but uses another gas?
We have MEAs in use over years which have probably been treated 50-80 times and they still work fine.
Thomas

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What is your settings

Post  brooks on Sat Jun 13, 2009 5:07 am

ThomasMCS wrote:Dear Steve,

we use a plasma cleaner from Harrick
http://www.harrickplasma.com/products_cleaners.php

I have no Idea about the plasma gun you are using.
Maybe you can try on an old MEA to see if our chips tolerate it.

Thomas

Thank you for providing these.
May I know the detail settings of the cleaner, like power settings(?? Watts)?

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Re: Coating Protocol

Post  Frank MCS on Sun Jun 14, 2009 7:51 pm

Dear Brooks,
for the Harrick Cleaner, we use the maximum setting, with plain air, for 1-3min.

Regards Frank
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Re: Coating Protocol

Post  brooks on Sun Jun 14, 2009 8:56 pm

Frank MCS wrote:Dear Brooks,
for the Harrick Cleaner, we use the maximum setting, with plain air, for 1-3min.

Regards Frank


Thank you!

I've check the website of your Cleaner, the "hi setting" of "PDC-32G/32G-2" is 18W, and for "PDC-001/002" is 29.6W.
Which model is yours? PDC-32G or PDC-001?

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PDC32G

Post  ThomasMCS on Mon Jun 15, 2009 5:28 pm

We use the PDC32G - just a somewhat older model...

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Re: Coating Protocol

Post  geissmbi on Wed Feb 17, 2010 11:43 pm

Hi,
I just started trying to culture primary mouse hippocampal neurons on standard MEAs. Until now, I have the same problem as you descibe: cell death and clumping of cells. I tried Poly-ornithine and laminin coating in various concentrations. Now I will try the PEI coating which is often mentioned in the forum. My question is know: How do you store the PEI soluition in Boarte Puffer? Is -20 degress ok? How long can one use the aliquots? And the Boarte Buffer can be stored at room temperature?
Thanks for your help!
Best regards
Maren

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PEI

Post  ThomasMCS on Mon Mar 08, 2010 6:56 pm

Hi Maren,

the PEI coating is extremely cheap, so we have never been really systematically optimizing storage times etc.
However, as amking the Borate Buffer is a bit painfull we usually prepare enough for about 2-3 Month cell culture and freeze in 1,5mL eppendorff cups at -20C.

Neither the PEI nor the buffer are very temperature critical. So it can be stored for use of about a week at RT or in a 4C refrigerator.

Most critical is to ensure that the PEI is properly dissolved. It is very hard to pipet as it is very sticky and highly viscous. As it is clear it is tough tto see if it is really completely dissolved...

Greetings

Thomas

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Re: Coating Protocol

Post  geissmbi on Mon Mar 08, 2010 9:07 pm

Hi Thomas,
thanks for your helpfull advice.
I just tried the coating and decided to store the Borate Buffer (500ml) at 4 degress. The PEI dissolved (yes, it was really not so easy ;-) some "glue like" feeling) in Borate is stored in 2ml Alliquots at -20. I think this will be ok, as you mentioned, that both are not so temperature senistive.
Neurons seems to be quite happy, but our Laminin is maybe not the best, and some Neurons are still clumping :-( But much much better than on PORN or PDL.
Best regards
Maren

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