I had some old HUVEC (P8) on pnc-Si transwells and PET Costar transwells that I didn’t know what to do with. So I tried a permeability study to see if there were any unexpected roadblocks in the protocol. I seeded HUVEC @ 50,000 cells/cm2 on PET and pnc-Si and allowed them to grow for 6…
In my previous transport post here, I showed O-ring placement data which was motivated by my thinking about the unstirred water layer (UWL). I think it’s helpful to think about the total resistance to transport through transwell membranes as a number of resistors in series. There is an UWL in the donor well, then the…
After diffusing through a polymeric or pnc-Si membrane in the transwell format, diffusing molecules meet an “unstirrred water layer = UWL” on the bottom of the membrane. The UWL acts as another resistance to transport into the receiver compartment. According to some PAMPA papers, the UWL can be greater than 1 mm in thickness without…
A couple of weeks ago, I posted a discoloration array experiment that showed monotonic discoloration patterns here. This was a result that we’d been expecting for several months, but we never got it with NaCl and NaHCO3. Simplifying the solutions to NaCl and NaOH was the key. These results spurred some discussion on the mechanism…
The last few Tecan posts have dealt with adjusting gain values to optimize fluorescent standard curves. We now have a general protocol here, and a final standard curve for fluorescein. Here is the background-reduced standard curve for sodium fluorescein (Na-F) that Christine and I obtained. This was done with the gain set to 0.000125%, and…
We now have a linear standard curve for sodium fluorescein (Na-F) in clear 24-well plates. What is the detection limit? The detection limit is defined as the lowest background-corrected concentration that is 3 standard deviations above the noise of the background. The background here is HBSS with 10mM HEPES. I measured the dilutions a four…
Now that we have preliminary transport data from the Tecan, we need standard curves in order to get permeability coefficients. This is really tricky because the RFU (relative fluorescent units) are arbitrary and based on the Tecan gain. For example, a solution of 10uM rhodamine might be 5000 RFU with a gain of 80 but…
All of this talk about Zeta Potentials, electro-osmosis and electric double layers got me thinking about these phenomenon in relation to discoloration. We’ve known that salts accelerate discoloration, and we know that bicarbonate solutions accelerate discoloration as well. I explained the bicarbonate issue based on the equilibrium between bicarbonate ions and hydroxyl ions (pH). We…
This post is a continuation of the experiments posted a couple of days ago. In that post, I concluded that absorbance measurements of molecules transported through transwells were not possible if the transwell insert was kept in the 24-well during measurement. This was due to absorbance by the outer walls of the 24-well plate and…
Last week, Dave carbonized some w501 samples for me so that I could look into discoloration with this new type of silicon stabilization. The carbonization recipe was: – 1 min 50:1 HF strip to form Si:H – Purge RTP with 100% Ar for 5 minutes – 10 min 95% Ar 5% C2H2 flush @ 200…