My goal for this experiment was to see whether vacuoles form in primary cells. If they did, this makes the case that the results with bEnd3 are more physiologically relevant since primary cells are better models of in vivo behavior. Here, I simply seeded P8 HUVEC at 50000 cells/cm2 on plastic wells, PET and pnc-Si…
I wanted to confirm my results from this previous post. Here, I used lower concentrations of bafilomycin (10, 25 and 50nM) in growth media. Everything else was the same (except the cells were 1 passage older). The results are the same-lots of vacuoles in the control sample and no vacuoles at all in any of…
I’ve been trying to convince myself that the black holes seen in endothelial cells that we’ve been calling vacuoles are actually vacuoles. The first set of experiments involved staining cells with TAMRA, a fluid-phase marker which should get pinocytosed into vacuoles. Although there has been co-localization between ‘black spots’ in the calcein channel and red…
Chris asked me to do some scans of samples last week but I didn’t have time to get to them. I was able to scan them today since I was setting the AFM up for my class anyway. I took 1um square scans (512×512) at 1Hz for all samples. The target and set point voltages…
Last week, I finished up an initial experiment to investigate whether pnc-Si membrane geometry affects cell morphology. To do this, I used a mask with 2×2 arrays of different size squares and also samples with 4 slits of different lengths and widths. A lot of the samples broke before I got the wafer, so this…
Another one of the questions I’ve been trying to answer with regard to endothelial cell vacuoles is whether membrane mechanics or permeability drive vacuole expression. I’ve thought of several experimental designs to test this that haven’t worked (i.e., using very small volumes on the apical side to minimize permeability, using PDMS plugs to block the…
One of the ongoing efforts with endothelial cell culture on pnc-Si is to confirm that the holes in these cells are actually vacuoles. In my last post, I tried to do this with FITC-Dextran and TMRIA with indefinite results. I ordered some 5-6-carboxytetramethylrhodamine (TAMRA, C-300 from Invitrogen) to do this test again. This rhodamine dye…
Tom recently designed a wafer with 1cm2 chips containing a 2×2 array of 1mm2 square membrane windows that fit on 384-well plates. I wanted one of these chips to get a preliminary data image for my R21 application. Instead, I saw something much cooler. I plated bEnd3 cells on the pnc-Si side of this chip…
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…