This post is related to the HIV drug screening project that Carrie has been working on. To refresh everyone’s memory, the aim of this project is to use pnc-Si as a filter to separate high molecular mass (HMM) protein-RNA complexes from smaller protein monomers/multimers. Small molecules that break down the HMM form of this protein…
This is simply the latest repeat of a blood-brain barrier co-culture on PET and pnc-Si transwells. These are P17 bEnd and P24 NG10815, each seeded at 50000 cells/cm2. As usual, I seeded bEnd on the pnc-Si side, upside down, and NG10815 on the well side, facing up. I took TEER measurements every other day for…
In most of the images I’ve posted of bEnd3 on pnc-Si, it seemed like there were more cells crowded onto the free-standing pnc-Si compared to supported pnc-Si. This post shows that this is indeed true. To do cell counts, I stained cells with Hoechst 33342 (1uM in media) for 15 minutes @ room temperature and…
All of my recent posts on VEGF, VEGFR2 and NF-H imunofluorescence have included data from cells grown on coverslips only. This was my first attempt to try to do immunofluorescence staining on intact pnc-Si transwells and then pop the chips out of the transwell plastic in order to image at 100X. Since VEGF is involved…
In order to validate the salt diffusion test I just posted, I’ve been measuring H2O2 diffusion with SC348 and the Amplex Red Assay from Invitrogen. I first showed that H2O2 (=34 Da) passed SC348 in this experiment. Here, I just repeated with slightly different standard curves and positive (‘regular’ Sepcon, SC195 = 4% and 13.9nm)…
About a month ago, I posted some preliminary data that showed NaCl diffusion through SC348. This was motivated by my observation of vacuole formation on SC348. Since my hypothesis is that cell culture substrate permeability is the key factor for vacuole formation, cells shouldn’t have formed vacuoles on SC348. Unless it is actually porous. This…
For my BBB project, I’ve wanted to get nice images of glial cells and endothelial cells on opposite sides of pnc-Si membranes to demonstrate 1) optical transparency 2) that both cell types are in focus (and thus right on top of each other). I’ve gotten some images like that in phase contrast but I also…
I few weeks ago, I had a couple wafers made with different size squares and slits. I posted my first results here and here. In that experiment, I showed pretty dramatic 2D structures over both supported and free-standing membranes with rectangle shapes. Also, I showed vacuole formation at (almost) the single cell level on square…
Vascular endothelial growth factor (VEGF) is one of the important growth factors/signaling factors that affect endothelial cells. It is involved in angiogenesis, vessel regrowth during wound healing, vessel development and other vascular functions. Since this is the most commonly studied growth factor in in vitro angiogenesis assays, I’ve been thinking about it for awhile. If…
In all of my experiments involving vacuoles, I’ve seeded at 50000 cells/cm2 which gets the cells to confluence after ~ 1 day of growth. I’ve always seen vacuoles with these parameters. For this experiment, I wanted to see what the cells would look like after 1 day but at a much lower seeding density. The…