C2C12 Cell detachment from 96 well plate

Hi Pillai,

Thank you for contacting Cell Analysis Technical Support.  Have you tried Matrigel or Cell Tak at all?  I recommend trying one of these as they tend to be stronger adhesives.  

Thank you and please let me know if this helps!

Courtney 

Courtney Nadeau Watts

Technical Support Scientist/Remote Engineer

Cell Analysis Products

Phone Contact: 800-227-9770 (Option 3 , option 8)

Email Contact: cellanalysis.support@agilent.com

I understand the use of Matrigel and cell tak, but we don't use it because laminin facillitates better differentiation. But apart from that, the problem I am facing is that the cells detach after I have switched to Seahorse assay medium DMEM pH adjusted to 7.4. The cells are attached uptil that point.

Hi Pillai,

Thank you for the additional clarification. I found this differentiation protocol using Matrigel, just in case you wanted to take a look at it. I have a few follow up questions:

1. Can you tell me how many hours after differentiation the cells are being washed with the Seahorse media?

2. How many cells/well are seeded?

3. When washing, are you adding the media to the side of well rather than directly on top of the monolayer?

4. How much media (uL) are you leaving behind when washing the plate?

Thank you,

Courtney

Hi Pillai,

Thank you for the additional clarification. I found this differentiation protocol using Matrigel, just in case you wanted to take a look at it. I have a few follow up questions:

1. Can you tell me how many hours after differentiation the cells are being washed with the Seahorse media?

2. How many cells/well are seeded?

3. When washing, are you adding the media to the side of well rather than directly on top of the monolayer?

4. How much media (uL) are you leaving behind when washing the plate?

Thank you,

Courtney

Hi Courtney,

1. I differentiate for 5 days and change the media on the 6th day.

2. I seed roughly 2000 cells / well.

3. I add / remove the media from the side wall.

4. Rougly 20µl of media is left behind.

Thank You,

Pillai

Hi Pillai,

Thank you for this information. I recommend trying a different cellular adhesive to see if this improves the detaching.  Additionally, you may want to increase your seeding density as 10K/well was used in this paper (though these cells were not differentiated).

Thank you and please let me know if using a different cellular adhesive shows any improvement!

Courtney