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…
We have a high school student, Nikita, (from my Summer Nanotech class) who is volunteering in the lab. Jim and I have assigned him the project of exploring diffusion of very small molecules through porous and putatively nonporous membranes. Currently, we are focusing on hydrogen peroxide (H2O2 = 34Da) and detecting it with the Amplex…
In this experiment, I wanted to test how quickly vacuoles form in endothelial cells when cultured on pnc-Si transwells. Here I used 30nm SC502 RTP’ed samples with the pnc-Si side down. I seeded P9 bEnd3 cells at 50000 cells/cm2 on the pnc-Si side, allowed the cells to attach for 2 hours and then inverted the…
As I was preparing for the BMES conference, I took a few pictures of endothelial cells on pnc-Si transwells that showed extensive vacuole/endosome expression (here). The most dramatic example occurred on a 50nm-thick membrane that was barely porous. So I wondered – are cells responding to mechanical or porosity differences (or both, or something else)?…
In my 1st post detailing glial cells on pnc-Si transwells, I saw lots of cell clumps above the membrane window (see here). Since these cells were grown in the wells, I thought they could be piling on top of each other in the wells and getting trapped. Here, I decided to flip the sample so…
I’ve suspected that the barrier function of endothelial cells might be surface-dependent (that is, cells grown on the pnc-Si side vs. cells grown on the well side). I hypothesized that the interface between membrane and well walls would alter the continuity of the endothelial monolayer when cells are grown on the well wide. To test…
In my last couple of posts with respect to endothelial cell culture on pnc-Si transwells, I presented images of cell clumps over the active pnc-Si membrane area of transwells (here and here). There were dead cells associated with these clumps and this is a concern for barrier function experiments. This post shows a counter-example of…