For everyone conducting biological studies, this paper highlights how to think about sample size and replicates. It suggests using “SuperPlots”, which color codes data points from different experiments to better visualize the type of variability seen (i.e. random variability, experimental variability, etc…). SuperPlots: Communicating reproducibility and variability in cell biology Jim and I recommend everyone…
Introduction A recent collaborative publication from RIT developed a modular µSiM to address the lack of physiological fluid flow in conventional open-well devices (Figure 1). The magnetic latching assembly of this design allows for quick configuration into a fluidic setup. The flow module is a microfabricated PDMS insert that contains a channel over the membrane…
Introduction: Many applications of the µSiM require fluid to be continuously supplied to the microfluidic devices. We use laboratory tubing (typically Cole-Parmer/Tygon Tubing with I.D = 1/16″) that is connected to syringes or peristaltic pumps to do so. However, interfacing the tubing with the current entry and exit ports for the bottom channel is not…
Introduction The basement membrane (BM) is a critical component of a healthy blood-brain barrier (BBB) model. Further, the BM breaks down in several diseases. Both pericytes and endothelial cells can contribute to basement membrane production. This post will explore a method to localize basement membrane components – collagen type IV (Col IV), fibronectin (FN), and…
The idea of adding a pulldown modification to our bacteria assay protocol came about after we started to believe that pore discovery was our rate limiting step of the transmission process, discussed here. Initially, it seemed to benefit our assay as it bumped our performance up significantly. Upon closer inspection we realized that we had…
On our quest to optimize bacteria assays using µSiM-CAs, we recently discovered that higher initial bacterial ODs decrease transmission in positive controls, described here. This was something we did not initially anticipate, engendering a new hypothesis around the idea that less initial bacteria leads to more transmission across our membranes in the µSiM-CAs due to…
After titrating our pulldown volume to avoid pulling Staphylococcus aureus through our membranes, discussed here, we found that 10 µL withdrawals allow us to continue to use the pulldown modification without compromising our results by providing false positive transmissions. With newfound confidence in this pulldown modification, we took a step backwards to repeat a bacterial…
On our quest to improve the performance of bacterial assays that use the µSiM-CA by modifying our standardized protocol, our most recent modification has caused controversy. Described here, we recently decided to add a step to our protocol where we pull fluorescently tagged Staphylococcus aureus onto the membrane by withdrawing a bacteria filled solution which we load…
For the osteomyelitis project which I have been working on for quite some time now, my collaborators and I have been working solely with 0.5 µm microporous membranes. To ensure that every membrane we work with has pores of the right size and are defect free, the two following protocols were created. The first checks…
The last time I did live imaging on the confocal microscope was in December of 2019. Then I was imaging the capture of 1.24 µm diameter beads in 1 µm slit pore membranes to visualize the difference between steric and affinity based capture. This time around, all of the materials are different, but the process…