PDMS/membrane deflection was measured on Veeco profiler by JP and me. The main problem with this kind of measurement is that it is taken form the backside of the membrane and the walls need to be excluded from the measurement. Using 20x objective helps but still has some problems. PDMS was first spin-coated on the…

In my last post, I concluded that switching the o-ring position from below the membrane to the top in the sepcon produced no leaks. The reason for this switch was to reduce the 1mm “dead-space” (created by the o-ring) that adds to the unstirred water layer (UWL). The next thing that Barrett and I looked…

PES Membranes: I tested 100 and 300kD PES membranes from Pall. 5 and 20 nm particles were used in both water and 10mM KCl. I won’t show all of the data, but I didn’t see any particles in any of the filtrates by absorbance or DLS. The filters were also stained red after the experiments….

In these trials, I used TER to look at the resistance between membranes with different amounts of pinholes. Finding a trend would produce a quicker way to scan for pinholes as opposed to checking each slot on the wafer with the microscope. The table below is my raw data. Wafer Number of Pinholes Resistance (ohms)…

The new electroosmosis wafers have randomly distributed chips with 1, 3, 6, or 9 .1x.1mm freestanding pnc-Si squares.  The idea is that we can use these to get a better EO rate vs. active area curve.  SC125 seemed to have some sort of film on it, but I decided to give it a shot anyhow…

In my last post on HUVEC intercellular junctions, I just showed cadherin staining.  I repeated this protocol but also tried immunofluorescence for ZO-1, the tight junction protein. These are HUVEC P7, 6 days post-seeding @ 50,000 cells/cm2.  I looked at glass coverslips, pnc-Si and commercial PET transwells.  Since background fluorescence was so high in my…

The BT method mentioned in this post seemed to give stable particles, but the BSA-Au did not work in separations.  After about 24 hours, the particles retained pink color but seemed to settle in tubes.  This may be due to reversable flocculation, not aggregation.  The retentate and filtrates in separation experiments both lost all color,…

One of the markers of endothelial monolayer maturation/barrier function is the expression of adherens junction and tight junction proteins.  The adherens junction is composed of transmembrane proteins called cadherins (among other proteins) that link cadherins of neighboring cells to the actin cytoskeleton.  They are found in most (all?) vertebrates and are necessary for cell-cell adhesion…

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…