In this post I will describe the use of the 3-D computational diffusion model as applied to separations with thick membranes. Experimental: Track etched membranes were trimmed to fit into round plastic sepcons and were sealed in the traditional way using an o-ring. 20uL of protein sample was pipetted inside the sepcon and 20uL of…
My previous 2 posts on co-cultures presented images but I also tracked the TEER in these experiments. This post shows that data. The top 2 graphs are from the 1st co-culture experiment – the bottom graph is from the second experiment (where my PET controls died), as % Day 0: On PET, the coculture TEER…
In my last post (the first co-culture), I showed that bEnd3 cells exhibited a different morphology when they were co-cultured. I also decided that I would reverse the orientation of the co-culture in order to better visualize the endothelial barrier. That is, grow bEnd3 on the basolateral side and NG10815 on the apical side of…
This post presents cell images from the first successful co-culture experiment. I seeded P8 bEnd3 cells alone, P13 NG108-15 cells alone and both cells on PET and SC500 pnc-Si transwells. For this experiment, I seeded 50,000 bEnd3/cm2 on the apical side and 50000 NG108-15/cm2 on the basolateral side of the transwell. I tracked TEER (future…
One of the themes in my recent posts has been the presence of ‘vacuoles’ over free-standing pnc-Si and PET membranes. We’ve assumed these are vacuoles because endothelial cells form vacuoles, they don’t stain with live cell stains (and so have no cytoplasmic enzymes) and there can be several of these structures in each cell. In…
I’ve been wondering if there were differences in actin/cytoskeletal structure of cells on supported vs. free-standing pnc-Si. A couple weeks ago, I had a couple of extra pnc-Si transwells so I stained for actin. I followed the actin staining (rhodamine-phalloidin) method I uploaded to the protocols section of the lab, and I counterstained the nuclei…
A couple weeks ago, I showed some preliminary data on H2O2 detection via the Amplex Red Assay, which is a project for one of our high school students. This generated some discussion at the last NRG meeting about diffusion through “nonporous” membranes. Jess wanted to look at very small molecule diffusion in her set-up so…
In the past couple of weeks I’ve tried a few more molecules to flesh out the experimental verification of the separations model. This is a quick update of the results. In the following figure, the blue line is the predicted sieving curve, the green line sieving given a no membrane/free diffusion situation, and the blue…
One of the things that I’ve been working on recently is trying to explain the extreme elasticity of the pnc-Si material which is connected to its nanocrystalline structure. There are two parts in this question. First, how are amorphous or crystalline phases involved during stretching? Second, does the crystalline phase change under application of mechanical…
One of the features of the gold nanoparticle separations in SepCons that was under-appreciated by the reviewers at Science was the fact that we could get very high sieving coefficients in the neighborhood of the cut-off. For 5nm gold the sieving coefficient was 0.8 (i.e. the filtrate was 80% as concentrated as the stock solution)…