A couple months ago, I tried to use tetramethylrhodamine (TAMRA) as a fluid-phase marker to label vacuoles. The idea is that the only way this stain could be internalized is if it is pinocytosed, eventually ending up in vacuoles. If you follow this link, the dye worked OK but not great. I found a couple…
It’s been a while since I’ve done long-term BBB co-culture experiments on transwells but I recently completed a study. I was particularly interested to determine whether I could reproduce this spike in TEER that I measured in 1 previous experiment. I also needed repeats anyway. I followed the normal procedure – seeded P11 bEnd3 on…
Last time I cultured fibroblasts on pnc-Si transwells, the cells got super-confluent over the membrane and made the analysis difficult. Basically, I wanted to see if fibroblasts formed vacuoles/2D structures over free-standing membranes. I thought the multi-layer cells could have choked off permeability and prevented the higher layers of cells from even sensing that they…
Along with differently sized square membranes, I also designed a wafer with different sizes of slits. On each chip, there is a 4000, 2000, 1000 and 500um long window. The thicknesses of these windows are 200, 100, 50 and 25um. I’m interested to see if there is an aspect ratio that causes endothelial cell alignment/tubulogenesis. …
In my last post on this subject, I showed that cutting off permeability of pnc-Si chips inhibits vacuole formation in endothelial cells. To do this, I seeded cells on the membrane side, allowed them to attach, inverted them onto a media bubble contained within a cloning ring, wet the well-side with media and then placed…
One of the hidden secrets of endothelial angiogenesis assays on Matrigel is that non-endothelial cell types also form 2D capillary-like structures (tubes) on those gels. Motivated by this observation, I wanted to look at fibroblast behavior on pnc-Si transwells, specifically with respect to whether or not they formed vacuoles. Just to clarify – although Anant…
There’s been lots of discussion about “non-porous” membranes and the possibility that they are actually porous. By “non-porous”, I mean that a TEM of that membrane doesn’t have obvious pores by pore processing/our eyes. This could mean a couple things: either the pores are so small that they are hard to resolve by TEM or…
The other (see below) just-to-be-sure experiment I performed this week was to compare staining between 2 different live cell dyes. I typically use calcein AM to stain live cells green. This is a membrane-permeant dye which, once inside cells, is hydrolyzed by intracellular esterases to make it fluoresce green. Another very common live cell stain…
I recently started up another BBB co-cultures and I had a couple of leftover samples. I decided to use one for a ‘just-to-be-sure’ experiment since I realized that I never did a calcein-Hoechst 33342 co-stain. That is, I never used a nucleus-specific dye with calcein to confirm that the black holes (vacuoles) in the green…
I designed new masks with different sized squares and slits in order to study how the aspect ratio of free-standing membranes would affect endothelial cells. In this 1st experiment, I seeded bEnd at 50000 cells/cm2 and let them grow for 4 days. I then stained the cells with Live/Dead and overlaid the phase channel in…
