Some days in science, things just don’t go the way you plan. Today was a good example of that. You can have a very planned, thought-out experiment and then it all goes to **** (insert expletive of your choice).
We recently observed that an enzyme digestion kit that we were using was cleaving some of the antigens (cell surface markers) off our cells. In particular, CD4 and CD8, both of which are rather important for being able to identify T cells. Since we’re trying to establish whether a certain type of T cell is present in the brain when there’s a viral infection, these are, well, crucial.
The problem is that the enzymes are very good at chewing up cell debris and myelin and other brain-related gunk that just doesn’t separate out well otherwise, and we get a cleaner preparation. The bad thing is that if it’s trimming off CD4 and CD8, we can’t trust it to not remove other potentially important markers. Today’s experiment was to try to shorten the incubation times with the enzyme to limit the damage so that we could still get a clean preparation and less or no antigen (marker) loss.
After carefully setting up enzyme incubations of 20, 15, 10, 5, and 0 minutes (no enzyme digestion), we cultured half of the 20-minute cells to see if they would/could re-express some of those markers. Next, ALL the sample sets were separated by a centrifuge technique.
That was the idea, anyway. The reality: two of the tubes wedged against the post in the centrifuge. This prevented the buckets from swinging out all the way. The tubes had varying degrees of cell separation from “ewww, wtf is that?!” to “hey, that actually almost looks normal but really dirty.” It took 4 hours to get from start to total experiment fail. It actually was not the first time this happened, but the third.
There are these little rubber feet in the bottom of the centrifuge tube holders. These are important because the centrifuge is spinning these tubes at 1200 x g, and some tubes will break at that speed, especially without some cushioning. Your bones would probably break too. If we take them out, the standard-sized tubes fit but could potentially shatter under the force (thus spinning all our cells all over the inside of the centrifuge). If we leave them in, the tubes might wedge against the post again. We tried a slightly different (shorter) tube, but they were such a tight fit they were hard to get back out, which is no good either.
Sometimes experiments fail because you mess up. Sometimes they fail because your equipment messes up.
I am NOT doing the happy data dance today.