Should the sample be tumbling or stationary?

willadler

There was some recent discussion on ResearchGate about whether the sample should be stationary or tumbling between the electrodes. Dr. Renier wrote that the authors told him that it should be tumbling. It seems intuitive, though, that the brain should be stationary to allow complete electrophoresis, rather than the lipids repeatedly moving back into the center.

What has been everyone's experience with this? Does anyone have an idea why tumbling might be recommended?

joelrosiene

The only thing that I have encountered that might relate to this is that during my first run, one side of my brain cleared more thoroughly than the other. Moving tissue might mitigate this? I have also found, however, that if the current is too strong, and the tumbling too vigorous, that the gel and tissue hybrid begins to pick itself apart as it is dashed against the walls of it's holder.

Aaronoldre

Dr. Renier and the authors were both saying that they were running their pumps at 20 L/minute which I assume would cause their samples to tumble vigorously. @Joelrosiene I think you said you had your pump set to 3 ml/second. How did you decide on that speed?

joelrosiene

I use a fishtank circulator which puts out solution at about 10ml/sec or 600ml/min. This is much slower than the original speeds recommended by the authors. Even so, I found that a current of 10ml/sec directed straight at the tissue eventually caused the gel to break up after bouncing around inside it's holder. I used a short-circuit in my flow tubing to cut the speed down to about 3ml/sec, and everything seems to be much more stable now. I have also introduced a sort of break-water into the chamber, to break up the laminar flow of the current so that the tissue is not hit by such a strong current.

nicolasrenier

I'll know more about this issue in a couple of days (I'll be meeting with the Deisseroth team).

Even with the high flow rate, my sample is not bouncing around, because it is protected in the enclosure made with the cell strainer. It's just slowly turning around.

I did not experience any break down of the tissue during the ETC. I tried adult brains and spinal cords. After the hydrogel embedding, the tissue seems really tough.