Matt Boyle, Entry #2, Whats up Dog?

Fellow mad scientists--Whats up dogs? hope all is well in your labs!! I have been hard at work in the Mason lab contributing to a few different projects. 

Since I have told Dr. Mason about my interest in medicine, she has been so kind to let me spend (roughly) a day of each week in the clinic working with the dogs as they have blood drawn, and are either screened to be entered into one of her vaccination trials, or are actually hooked up to blood pressure/heart rate (EKG) monitors as well as fluid pumps and are administered a vaccine to treat their cancer.  This is probably my favorite part because I get to spend the day not only learning about hospitals and how they run, but I am also with dogs all day and despite them being very sick, and some even having a limb amputated (like Sally Belle below), they are always so happy!!

Sally Belle has osteosarcoma which is very rare as it usually only affects larger dogs.  She has had her rear right leg amputated but since recieving Dr. Mason's vaccine she has been doing extremely well.  She received the full course of  (three) vaccines and is now receiving her booster vaccines once every few months

While the hospital is great, nearly all of my time is spent in the lab.  From each dog that comes into to see Dr. Mason, we take a blood sample and more often than not, it is my job to work with these samples to extract what are called the PBMCs (peripheral blood mononuclear cells) which are essentially the lymphocytes (B Cells and T Cells) of the blood, among other things such as macrophages.  I have beaten this protocol to death over the last couple weeks but it is very cool and probably my favorite procedure here because you must pay meticulous attention to detail as you mix the blood with one liquid and then very carefully place that mixture on top of a liquid with a much higher viscosity than the blood mixture, forming two distinct layers.  These layers cannot mix as they are placed in the centrifuge for a half hour and most of the blood drops to the bottom while the PBMCs remain up top.  

PBMC Isolation -- The conical after being spun for 30 minutes at 1500 rpm.  The very small cloudy white layer that is above the clear layer which is above the red blood cells is the PBMC layer.  These are the cells we extract and culture before electroporating them with tumor RNA and injecting them back into the patient.

Another method we use here that essentially tells us whether or not we out experiments are working is called FlowCytometry.  After culturing the cells that we extracted from the dogs (the PBMC isolation), we stain the cells by permiabilizing the membranes of the cells and then adding antibodies to them.  For example, we add CD79a, an antibody that stains a protein on B cells, and when the Flow machine sends a laser through the cells, the ones that are positive for CD79a will glow and be plotted on a graph by a computer. This ultimately gives us the percentage of our cells that are B Cells. The same process is used to identify T-Cells, but with a different stain known as CD5. 

As you can see on the X axis, these cells have been stained for CD79a, an antibody that lets you see B Cells. You can see the large population to the right of the central axis, meaning that there is a large population that is CD79a positive, mening they are all B cells.  Anything to the left of that central line is negative for CD79a, meaning it does not express CD79a and therefore is not a B Cell

A significant part of my time has been spent doing research for the prospective horse vaccine that I introduced in my last post! (his name is Calvin).   I have had several meetings with Dr. Mason pertaining to the most effective and efficient way to put this vaccine together.  We are trying to pro-actively find the glitches that will occur as we carry over the dog protocols to a horse model.  To eliminate one of the variables, we were able to get the owner of the horse (who is understandably quite affluent if they want a vaccine for their personal riding horse...) to fork over a couple hundred bucks to purchase equine interleukin 4 which is a cytokine that promotes cell growth as it induces helper T Cells which in turn induces the creation of more (memory) B- Cells as well as cytotoxic (lethal) T Cells.  Cyclosporine is then added to the cells as it kills T Cells to ensure that no auto-immune events will occur as the cells are now outside of the organism (which would be the T cells killing the organism’s own B Cells…which is bad). Anywho, we have been working closely with New Bolton Center – Penn’s large animal campus – to get blood which should be here later this week! I will keep you posted as I aim to cure horse cancer!!