We’ve been getting some strange cell morphologies recently in short-term adhesion studies. My previous post on this subject links to a few of these results. Unfortunately, my last experiment into short-term cell morphologies on autoclaved and EtOH- sterilized samples wasn’t well-controlled since I didn’t have enough samples. Therefore, I repeated this adhesion study with proper…
Some transwell cell culture models require 1 week or more of cell growth in order to let the cells fully differentiate and form mature monolayer barriers. I had a couple of leftover samples a couple of weeks ago, so I measured the viability of cells on RTP’ed pnc-Si and PET transwells 7 days after plating…
Dave did a series of 20nm SiO2 film depositions at different temperatures (constant bias) and gave them to me to AFM. I guess he wanted to see if film roughness changed drastically over the range of ‘room temp – 400C’ in order to investigate this as a potential source of pinholes. I used a new…
Before Anant left for India, we were doing cell adhesion studies with pnc-Si transwells and getting some weird morphologies. Check it out here, here, here, here, here. This is in contrast with the 2D studies that Anant did for the cell culture paper in which cell adhesion looked good. The main difference is the transwell…
Earlier this week, Maryna and I did some AFM of her samples. She vapor-deposited parylene on the well-side of pnc-Si and was interested in whether/how well that polymer got through the pores. We got some really interesting-looking scans, but I’ll let her post those images. As controls, we looked at clean chips from the same…
One of Christine’s projects was to determine if pinholes affected electrical resistance (TEER) measurements. She summarized her work here. I collected her data and did some negative controls to finish this work up. Here’s the graph: The “no membranes” data is from Sepcon transwells with pnc-Si chips but without membranes (I broke them before assembling…
Christine and I have been looking into whether the O-ring position affects transport through pnc-Si transwells. The hypothesis was that the unstirred water layer under the Sepcon might be diminished by switching the O-ring from its normal position (“O-ring bottom”) at the bottom of the Sepcon to above pnc-Si (“O-ring top” or “pnc-Si bottom”). This…
Anant and I have been doing long-term discoloration experiments for the past few weeks. We set up samples in “ideal” conditions – pnc-Si in transwells with pnc-Si side down, autoclaved samples and in the incubator (lower OH-). The only non-ideal component is the use of DMEM+10%FBS with high NaHCO3 (3.7 mg/mL). Chips were RTP’ed at…
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…
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…