Matt Boyle, Entry #3, Working Like a Dog (or should I say horse)

The plan was to get samples of horse blood last Wednesday until Dr. Mason realized that we did not have enough irradiated k562 cells (irradiated means the membranes are sort of stuck in time but the cells are still alive) for the control of the experiment.  The problem was that irradiated K562 cells expressing CD40 Ligand had been in the liquid nitrogen for several months and when we irradiated the K562 cells we wanted to put them in the liquid nitrogen as well to make the conditions as similar as possible to the CD40L expressing cells; who knows the effects that freezing a group of cells down to          -150⁰ dos  to the viability of cells.  Therefore, we postponed the blood delivery to next Monday I had to go into the liquid nitrogen and thaw out a vial of K562’s, grow them to a total of three million, irradiate them, and freeze them down.  By the weekend, they were in the liquid nitrogen and had been in identical conditions to the CD40L expressing cells.

Speaking of the irradiator, going to use that machine is an intense process.  The irradiator is located in the basement of my building which also happens to be the rat facility (that you can smell every day from the stairwells).  Once you get downstairs you need to put on little boot things and a hairnet and gloves and then at another station you must put on a sterile gown that looks like you are scrubbing in to perform a surgery.  Then you must stand in front of a machine that does a retina scan that allows you to open the door with a separate key.  I told you it is intense.   Dr. Mason took me down once and I wasn’t even allowed in the room.  She scanned her retina, entered the room, and I had to watch from outside.

Dr Mason at the irradiator, in her sterile gown and hair net, as I look on through the small window in the door.

 On Monday morning, the blood of three horses was pulled at New Bolton Center and shipped to Dr. Mason’s lab.  Dr. Mason made sure it would be “hand delivered” as to ensure optimal condition of the blood.  The members of the lab that are participating in the horse lab (myself, Dr. Mason, lab tech Josephine, and PhD student Kazim) had a quick meeting to make sure everyone knew what their role was and how the bloods would be handled.

The three horses all had labels to identify them by (R600, R665, and R289) however their given names were also written on the tubes of blood – Wise guy, Class Fantacy (spelled like that), and Idol – which I thought was hilarious.

The vials in which the horse blood arived.  You can see that each horse had an ID number as well as a given name that was usually pretty funny.

I was in charge of extracting the PBMCs by using a protocol that I had discovered was successfully used in a lab in 1978 to extract equine PBMCs over ficoll.  We did not know if this 1978 procedure would be more effective than the protocol the lab uses for the dogs.  We had 20ml of blood from each horse so we used a single sample of 10ml to decide which protocol we would use.  We got decent yield of PBMCs from the 1978 protocol which was around three million.  We attempted PBMC isolation with another 10ml of blood, this time with Dr. Mason’s protocol, and got a yield of approx. 8 million cells and therefore used her protocol on the remaining 20 ml. 

The equine PBMCs are pretty visible -- they are the clear carpet-like layer right below the yellow (which is serum)

The PBMCs were then cultured under three conditions.  For each horse, a given number of their cells would be left alone to see if the B Cells proliferate on their own (obviously they will not, this is just a formality to say we evaluated this).  The next set of wells contains The PBMCs cultured with K562 feeder cells.  This is the control for the following row in which the PBMCs are cultured with K562 cells that express CD40 Ligand.  The only difference between the last two rows is the expression (or lack thereof) of CD40 Ligand on the surface of the feeder cells.  Therefore, if there is a difference in growth of B cells among these rows, it HAS to b

This is a diagram of the plate that the cells were cultured in.  For the three different horses, there were three different conditions that the PBMCs were cultured in (by themselves, with plain feeder cells, with feeder cells that express human CD4

We will check the cells on days 2,4, and 7 and add new growth media and cytokines so by next week

we will be able to stain flow them (mark with antibodies and computer analyze) to determine the content of all the wells.