Those with a great memory will remember that I’ve been struggling (1, 2, 3) to identify adherens and tight junction proteins (i.e., cadherins and ZO-1) in bEnd3 endothelial cells. Since I’ve recently moved over to investigate air-blood barrier co-culture models, I decided to see if my epithelial cells (RLE-6TN) express cadherins (they should since these…
Our collaboration with Alison Elder, et al. in the Dept. of Environmental Medicine has led us to talk about developing a co-culture model of the air-blood barrier. This barrier is ‘located’ at the alveolar sacs in the lungs, where lung epithelial cells in the alveolar space are exposed to air. These epithelial cells are separated…
I had some extra samples from last week and had been wanting to try SEM on cells, so I went for it this week. Specifically, I was hoping to get images of vacuoles in cells over free-standing pnc-Si. I followed a rather complicated protocol full of dangerous chemicals to fix the cells and then dehydrate…
I’ve been working recently with amyloid-beta (AB) provided to me by some people in Brad Nilsson’s lab. These experiments are related to the HMM diffusion experiments for our collaboration with Harold Smith. Basically, AB fibrils are very long (microns) and we are looking for ways to either prevent their aggregation from smaller peptides/oligos, or to…
One of the other suggestions from Angela Glading at lab meeting a while ago was to quantify the vacuoles on different culture areas. So, I selected images from 3 different experiments (all at day14 of growth) and counted the number of cells which expressed vacuoles on different areas. I then used my mask code in…
My last post dealing with this project was the first to definitively demonstrate HMM diffusion through pnc-Si. Unfortunately, there wasn’t a difference between the HMM filtrate signal for intact and digested (+RNAse) samples. As a reminder, HMM is a large protein multimer held together, partly, with RNA. We hope to show that HMM can not…
My last post was the first experiment I performed with rat lung epithelial cells on transwells. It seemed like there was vacuolization but it was delayed compared to endothelial cells (and only happened on 1 of 2 pnc-Si transwells). Here, I did a repeat with a lower seeding density (25000 cells/cm2) and with SC613. The…
A couple weeks ago when I presented at lab meeting, Angela Glading suggested that I look at epithelial cell vacuolization on transwells. This was in order to determine if vacuole formation was endothelial cell-specific. If I’m remembering correctly, the rationale was that the vacuole-less fibroblasts I showed were not contact-inhibited cells and thus not a…
Carrie and I have been trying to detect HMM (APOBEC3G) diffusion through Sepcons without much luck. We determined that the dilution factor (~1:30) for diffusion in our 384-well plate format was too high to detect HMM in the basolateral well. To try to get around this problem, I decreased the volume (and thus dilution factor)…
A couple weeks ago, I presented a potential figure for my paper that didn’t make sense. So, I did a couple more controls and put together this new figure. This is the “diffusion through ‘non-porous’ SC348/cut-off of nonporous membranes” figure. I redid it to include SC348 (the nonporous sample) and ‘typical’ pnc-Si as % of…