I'm trying to develop a reliable protocol for a caco-2 transport assay, but I'm encountering a problem with the TEER not being stable over the course of the assay.
I measure the TEER before I start the assay, after a 15 min wash in buffer, after I add the test compound, and after a 4-hour incubation before collecting the samples.
All my wells are seeded at the same time and have similar TEER values before I start the experiment, but over the course of it, some of them randomly drop by varying amounts. There doesn't seem to be a strong correlation between the drop and my treatment compounds. For each condition, some drop, and some don't, even with my controls. Sometimes it's an entire plate and sometimes it's one or two wells. I'll run a test of my conditions, and the TEER will be fine and then I'll do it again with cells of a similar age and passage number and all of the TEER values will be bad.
This is a huge problem because once the TEER falls past a certain point, the transport of my control (lucifer yellow) and my target start to exponentially increase, although at different rates, rendering the data useless. Given the time and cost of these experiments, I really can't afford this.
I'm fairly new to caco-2 cell culture. Does anyone have more experience and know what causes this or how to prevent it? The TEER doesn't fall all the way like it would if I were scraping the monolayer with the probe or something.
Conditions:
-Cells cultured on transwell inserts for 21 days, TEER around 1350 ohms/cm2.
-Culture media: DMEM +10% FBS, 1x antibiotic/antimycotic, changed twice a week
-Transport assay:
-rinse cells 1x with 1x HBSS +Ca, Mg
-Replace rinse with treatment buffer: 1x HBSS + Ca, Mg, 20 mM HEPES, 25 mM Glucose pH 7.0
-Remove buffer, add test compounds prepared in treatment buffer to apical wells
- place plate in receiver wells containing plain treatment buffer
-Incubate 4 hr at 37 degrees in a cell culture incubator
-Collect basal samples
*All test solutions are adjusted to pH 7.0 (due to different natural buffer capacities, this results in differences in salt concentrations but again, drops don't correlate strongly to any given test compound).
Yes. Your experience is quite real One of the frustrations of measuring TEER and relying upon it only for BBB integrity. I suggest you do not move the probes from cell to cell in a multi chamber system.
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