I am currently working on differentiating Caco-2 cells into enterocytes on 24-well Transwell inserts (0.3 cm² surface area). To assess their differentiation, I have been measuring transepithelial electrical resistance (TEER) every other day.
Despite trying different seeding densities, I am consistently obtaining low TEER values—with a maximum of ~30 Ω at 21 days post-seeding. This is much lower than the expected range for well-differentiated Caco-2 monolayers.
I am using DMEM-high glucose 4500mg/ml, with 10% FBS, 1% P/S, and maintaining the cells at 37°C with 5% CO₂. my cellls passages are > 20.
Has anyone encountered similar issues? Are there factors I might be overlooking, such as:
- Seeding density optimization?
- Media composition or supplements that improve tight junction formation?
- Incubation conditions or feeding schedule?
Any insights or suggestions would be greatly appreciated!
Hi Hala Altaher
- Media, supplements, and Incubation conditions are good.
- Correct cultivation requires appropriate materials and tools, As for the transwell on which the cells are grown, (Corning) brand was used, and it was very good, and it should be chosen with specifications related to the permeability experiment, such as (pore size: 0.4 micrometer) and other specifications you can find them in previous studies.
thank you for your reply Mohammad Hatem Rasras . I am currently using corning with pore size of 0.4 micrometer. still I am facing the difficulty of getting proper TEER values.
Hi
Now, you should try 3-4 different numbers (seeding density) and find the best one, because seeding density that are too high or too low will not forming the monolayer, and that will give a low TEER values.
I have another question: What about the protocol do you use to read the TEER values? can you write it?
Hello Mohammad Hatem Rasras I hope my reply is not too late!
I use the EVOM manual device, it is calibrated and shall be working well. I measure blank value (transwell with media, no cells) and then I measure TEER for each transwell 3 times (From the 3 different corners of the transwell) and take the average. I then multiply this to 0.3 which is the surface area for my transwells to acheive ohms per squared cm. this is my protocol. any advise would be appreciated! Best regards,
Hala Altaher
This is the same method I used in my previous research, but with different surface area.
Our Caco-2 cells: "Caco-2 cells were grown on several sterile 12-well plates with the following transwell specifications: 12 mm diameter, 0.4 µm pore size, polyester membrane, 1.12 cm² growth area (Corning, USA) at a seeding density of 50,000 cell/well, plates were then incubated at 37 °C, 5% CO2 and 95% humidity incubator."
However, make sure to use HBSS instead of (DMEM or PBS) to give a correct and accurate results only during TEER measurment.
If all else fails, test the cells in a transwell (1.12 cm² surface area) to see if the issue remains.
Good Luck!
Thank you, MohaRasras,atem Rasras for this tip. I actually just measure in the same media I grow my cells in (DMEM high glucose). Maybe that is affecting the readings? But again, I blank also using the same media! Do you use HBSS that is Ca and Mg free? Any recommended brands?
Hala Altaher
1) I used: Hank's Balanced Salt Solution (HBSS), PAN-Biotech, Germany.
- With Ca²⁺ and Mg²⁺
- Without phenol red
2) Yes, calculate TEER using HBSS for all transwells (control, treated, blank).
I calculated the blank transwell with HBSS using a 1.12 cm² growth area, and it gave a TEER value of 125 Ω·cm².
* Do not use DMEM (high glucose will affects on TEER value) or PBS.
I’ve attached a PDF file of the Caco-2 monolayer permeability protocol for more information.
Good Luck!
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