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…
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…
This was mentioned in the meeting today, and I’ll try to describe what’s going on here. We start out with a system with a smaller volume on the top of the membrane: If we start out with 10 mg/mL in the retentate, and assume perfect equilibrium of the protein/particle: The concentrations in each compartment would…
This experiment is to determine if the time between the SC1 clean and when the wafers get deposited matter. Currently there is suspicion that an SC1 from an RCA clean should at RIT should be done right before deposition, . So what is done typically is that lithography steps are done weekly at RIT to…
I ran new experiments over the weekend in which I tested NaCl at 10, 25, 60, and 155mM. I also ran filtrations in the presence of 10mM LiCl and 10mM CsCl. These experiments were performed with chips from wafer # 024. I am only providing the Filtrate / Retentate ratios. It is obvious that the…
I tried particle separations with the 30kD cellulose membranes from Millipore using the same setup as previous experiments. Basically the results are that neither 5 nor 20nm particles pass through the membranes whether in water or 10mM KCl. Absorbance Data: Sizing Data: No peaks were found in the nm range for any of the filtrates….