Caco-2 cell culture

It usually isn't necessary to add the thawed cells to neat FBS. The important thing is to get rid of the freezing medium. This can be accomplished by adding the thawed cells to 10 ml complete medium containing 10-20% FBS (depending on the cells), pelleting the cells by centrifugation, and then seeding them into complete medium in a dish or flask.

Original Message
From:Yvonne18
Date:03/28/2013 04:50:00
In response to your message from 3/28/2013 2:30:00 AM
Here is the details of Caco-2 cell line (Seed Details:- CACO-2, Accession No 86010202, Lot No. 10J006, Pass 42, date 01/11/2010)

If culturing from a cry-frozen batch of cells, take care to thaw the cells quickly, and immediately adding them to 10ml of fetal bovine serum and spinning them down (5min at 1500rpm). For the culture medium you could try adding HEPES buffer (25mM) to keep the pH stable and some protocols also prescribe adding human transferrin to the culture medium. Products like "glutaMax" could also help to stabilise the cell attachment and growth.

Hope this helps,
Best regards,

Leonie Vogt

Thanks Leonie Vogt,
I will try this

In response to your message from 3/28/2013 2:30:00 AM
Here is the details of Caco-2 cell line (Seed Details:- CACO-2, Accession No 86010202, Lot No. 10J006, Pass 42, date 01/11/2010)

If culturing from a cry-frozen batch of cells, take care to thaw the cells quickly, and immediately adding them to 10ml of fetal bovine serum and spinning them down (5min at 1500rpm). For the culture medium you could try adding HEPES buffer (25mM) to keep the pH stable and some protocols also prescribe adding human transferrin to the culture medium. Products like "glutaMax" could also help to stabilise the cell attachment and growth.

Hope this helps,
Best regards,

Leonie Vogt

Here is the details of Caco-2 cell line (Seed Details:- CACO-2, Accession No 86010202, Lot No. 10J006, Pass 42, date 01/11/2010)

For the 500 ml Complete EMEM Medium preparation in EB:- Incomplete EMEM-440 ml(pH-7.41), pen/step-1%- 5ml, 10% FBS- 50ml, 1% NEAA- 5ml used.
The Complete EMEM medium warm around one hour in water bath before the use of the cells.
There was only about 5% attachment of cells was found on next day . changed fresh medium after 2 days . found 3-4 attached cells but not true attached they slowly disappeared and many dead cells in the base of T25.
anyone can suggest me way of overcome this problem

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Original Message
From:luo142
Date:10/08/2012 13:54:00
Thank you so much. I have another concern: I am also wondering that the HBSS is commonly used for cellular uptake of caco-2 cells, but I assume that it should be Ok to use DMEM, too? Could you please share your opinion, or some reference on this? Thank you so much!

Please see my last post. It answers your question and provides a reference.

Thank you so much. I have another concern: I am also wondering that the HBSS is commonly used for cellular uptake of caco-2 cells, but I assume that it should be Ok to use DMEM, too? Could you please share your opinion, or some reference on this? Thank you so much!

DMEM is fine to use for transport studies. The article at this Link is an excellent methods paper that uses DMEM for transport experiments.

If you can't access the article, post your email address and I will send you the pdf.

Hi,

I am currently doing Caco-2 cell monolayer transport experiment. However, my samples (protein solution) will precipitate in HBSS buffer but can suspend well in DMEM. Can I use DMEM for transport study? Thanks.

Best,
Yangchao

TEER values at two different times, and after a medium change, will frequently be different. Several things can cause TEER values to vary, such as temperature, ionic strength of the medium or buffer, other medium or buffer constituents, location of the electrodes, cleanliness of the electrodes etc. To get exactly the same TEER values before and after a procedure, everything has to be the same. Try taking a reading, aspirate the medium, replace with PBS or Hanks buffered salt solution, aspirate, replace with medium, read again. Chances are the readings will be different. Even though you read the resistance both times in medium, subtle changes could alter the reading. One thing to be aware of is residual PBS or Hanks on the cells before adding medium for the second reading. The usefulness of the TEER value is in showing changes in permeability throughout the course of an experiment. Not necessarily before and after an experiment.

One question: How long does the TEER value remain depressed after an experiment? Does the TEER increase gradually?

Hello everybody.
When I am performing my Caco-2 assays I always observe a drop in TEER value after the experiment. The TEER before the experiment is always between 300 and 400 ohmcm2. After the experiment I remove the test substances and apply cell culture media, and the TEER is then always around 200-300 ohmcm2. Has anybody experienced this before?? This also happens with well-known non-toxic substances like lucifer yellow, in tested concentrations which should not affect the cell viability.
I am really thankful for any help you can provide!

I had the same problem. Now I use a high dose of glutamine (6mM final) that help cells to attach after defrosting and grow very well.

There are a number of things that can result in a rise in TEER values. One is an increase in cell volume, which will decrease the space between cells. Also, some transport inhibitors can increase the TEER. Here are some links:
Link
Link
Link

Post your email address if you need pdfs.

Did anybody observed rise in TEER values from pre to post experiment (this couldnt be solubility issue as compounds are highly soluble)

Did anybody observed rise in TEER values from pre experminet to post experiment, as for some compounds i am seeing rise in TEER values in post experiment (this is not solubility problem of compound)

Did anybody observed rise in TEER values from pre experminet to post experiment, as for some compounds i am seeing rise in TEER values in post experiment (this is not solubility problem of compound)

Dear BlueDevil,
Yes I agree. I will look into it more :)
Many thanks!
Regards,
Kam

Good luck. Anything you find out could be very helpful to everyone that reads this thread. Post it if you have a chance.

Dear BlueDevil,

Yes I agree. I will look into it more :)

Many thanks!

Regards,
Kam