Hello, I'm James Volta with the Exercise Science Program in the Department of Kinesiology Recreation and Sport at Western Kentucky University. And what we're gonna do is we're gonna show you the procedure that we have developed and the procedure that we use in order to measure exercise in dues, lymphocyte, apoptosis. So let's go take a look.
A finger stick blood sample is obtained using universal precautions finger drop. The fingertip is sterilized, then punctured. The first drop of blood is wiped away, and then the blood is drawn into capillary tubes or micro beds.
10 microliters of whole blood has pipetted into an antibody panel containing flow cytometry, standing buffer appropriate fluorochromes, including annex and V seven amino actin D, either CD four, CD eight, or CD 19 and CD 45 ra in addition to a cells only tube, we have used compensation beads as a control to help set up our experiments. The sample is thoroughly mixed in vortex and then allowed to incubate a room temperature in the dark for 30 minutes. Following incubation samples are centrifuge for 10 minutes, and then the samples are decanted in one smooth motion.
300 microliters of red blood cell lysis buffer is added and the samples are thoroughly vortexed. It is very important that the sample and the lysis buffer are thoroughly mixed. To facilitate maximal red blood cell removal, you'll notice that your sample begins cloudy.
But after at least 15 minutes of incubation at room temperature, it will clear as red blood cells are Ls, 300 microliters of phosphate buffered saline is added acting as a stop solution to end the lysis reaction. And samples are again centrifuge for 10 minutes. Note that following the centrifusion procedure, you'll have a pelle of cells on the bottom of your tube decant in one smooth motion, thoroughly vortex to resuspend cells and solution.
And your sample is ready to be analyzed by flow cytometry. Here we show representative forward inside scatter plots gating into lymphocyte subfractions, and the identification of anex and V positive cells within the subset. So we've just described the methodology that we use to determine exercise-induced lymphocyte ptosis, and we think that there are a lot of further applications that can be used with this methodology.
It can really be used to look at any type of white blood cell besides lymphocytes and and lymphocytes, subfraction. And it can also be used to measure any marker that is found on the outside of the cell. So we really think this has some greater application.
