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Posts Tagged ‘experimenting on animals’

It’s not often that i work long hours in lab.  I usually come in to do what i need to get done, then go do something like walk the dog or tend the garden or go hiking.  Sometimes i read articles in the backyard.  It is apparent to me and to most people i work that i do not do as much work as i could.  But, i am happy not working at break-neck speed so that i may enjoy other realms of life a bit more.  My PI may not be too happy with this, but he’s exactly the same way and knows it.  Brags about it, even.  But then, he probably worked pretty hard in grad school… still, i’m secretly hoping that one day he will tell me he was a slow poke too.

So, 8 straight hours of work in lab will tire me out.  I’ll wonder how people in real life can do it 5 days a week.  But then, seemingly out of nowhere, i will muster a stupendous amount of energy and stamina and complete a  16 to 18 hour experiment that requires almost constant work-  every time i stop to pee or stuff food in my face i feel like i’m letting the quality of my data decline.  Luckily i am able to recruit people to help me- the experiments would be impossible without their help (ie, all the cells would croak and i’d get shit for data and there aren’t enough hours in a day), and i just wouldn’t survive mentally or physically without their support.  I learned this craziness from Jess- i used to think her 20 hour experiments (into which i put my share of grunt work) were pure insanity.  How could she possibly stay up into the wee hours of the morn running the flow cytometer?  But i get it now.  Especially the part about supplying snacks for your helpers.

Anyway, i”ll start with yesterday’s preparation for today.  It involved mixing up several liters of buffer and media; making 8-color fluorochrome-tagged antibody cocktails; mixing cell separation gradients to the correct percentage and aliquoting to tubes; prepping solutions – formadehyde, sugar water- and tubes in which to collect bits of liver to section and stain for beautious fixed and fresh-frozen immuno-histochemistry pictures; wrapping and autoclaving several dissection kits- scissors, blunt forceps, pointy forceps; collecting all the crap that i needed to bring down to the vivarium- like pipetmen, pipets, slides, tube racks, petri dishes, etc- and putting it on a big cart (the sight of which always makes the vivarium staffs’ eyeballs pop out because they know it’s going to be a bloody, stinky, sweaty mess down there for 4-5 hours); labeling no fewer than 263 tubes; then biking up Capital Hill at 8pm to have a Molly Moon’s sundae for dinner in celebration of Goo’s birthday.

I am slowly, slowly learning to prepare for experiments (especially big ones) before the day of.  Now, by the end of my third year in school, i seem to be doing ok.  I am at least less of a pain in the ass to the people helping me with the experiment and to my own self.   So, as i sit here at the flow cytometer at 9:30pm on the day of the experiment, it is not because i was under prepared and had to scramble around making buffer and labeling tubes this morning, it’s because this is a big fucking experiment.  I mean, i stopped at PCC to buy bagels for my helpers on my way in and was still here, showered,  in lab by 8am.  Granted, i could always prepare more.  More reading and research into the best techniques, or looking at past notes wouldn’t hurt.  I just realized that i forgot to stain my cells for an activation marker that’s fairly common in these types of experiments.  Whoops.

I should mention at some point that this experiment is aimed at measuring/evaluating/figuring out what the heck is going on during T cell priming against immunization with genetically attenuated malaria parasites. In mice.  Who get injected with transgenic T cells.  That are green.

This morning i biked in and before i showered, got the collagenase buffer out of the fridge to start warming up in the water bath so that the soon-to-be-added collagenase would dissolve.  Then i went downstairs to shower.  I did not wash my hair, because it always gets nasty and matted down by 5 hours in a vivarium hair net anyway…  but now, at the end of the day it’s especially gross-tastic.  I finished my day-of preparations which included weighing out and adding collagenase to the buffer, grabbing a tube of  mouse anesthesia,  filling two ice buckets with ice, and getting all of my collection tubes out of the fridge/off the bench and onto the cart.

8:45 I headed downstairs to the vivarium, where i was met by my new malaria buddy, Jess #2, and we transferred all the stuff from the lab cart onto a clean vivarium cart.  We went through the vivarium barrier (read “super clean area for immunologically messed-up mice”), which involves putting on a second layer of booties, gloves, a humongous gown (hence why we get all sweaty),  a face mask (which i just wear under my chin because it drives me crazy) and a hair net.  We set up the work side of one mouse room with a liver-perfusion station, a liver smushing station, and a lymph node and spleen smushing station.   Each gets a dissection kit, pipets and tubes and filters, slides, petri dishes, etc.  I brought four cages of mice (18 total) around on a cart just outside the door because we had occupied every other flat surface in the room.

9:20 Isaac joined us (he has a new baby so he doesn’t have to be on time anymore) and we gave Jess the run down on what was about to happen… for the next 4.5 hours.  I anesthetized the first mouse, perfused the liver (a technique that requires straining your eyes to find and insert a needle into the portal vein, all the while hunching over an open mouse with an amazing crick in your neck and upper back, and cramping up your needle-holding hand while trying to remain very still), handed off the liver to Jess and the spleen and lymph nodes to Isaac.  All of these are smushed up into single-cell suspensions via being pushed through a mesh strainer or rubbed between two frosted (rough) glass slides.   The liver cells are spun down to get rid of the hepatocytes, and the lymphocytes in the supernatant are taken off into a new tube.  All of the cell suspensions get filtered and go into a huge tray of ice.  Isaac and i switched off between perfusing livers and processing the spleens and lymph nodes.  When Isaac perfuses, he can collect blood after he snips the portal vein, then cut and remove a lobe of the liver with one hand while he holds the perfusion catheter with the other.  So for all the mice Isaac perfused, i have blood and tissue samples as well as spleen, lymph node, and liver cells.  I have not yet learned how to perform such acrobatics.

This disassembly line (heh heh) went on until about 2pm accompanied by discussions of running, frisbee, babies  and the birthing process and how much pampers suck, Jess’s upcoming wedding, and how much PIs gossip.  And occasionally the experiment or techniques at hand.  When we had finished the 18th mouse, we cleaned up the aftermath, peeled off our now nasty gowns, gloves, and hairnets, wheeled the cart of cells to the elevator, and booked it to the lunch room for bagels and cream cheese.  It’s gross to get hungry while perfusing mouse livers, but it can’t be helped.  Isaac swears he’s found the perfect timing and dose of coffee in the morning to suppress his appetite but allow him to hold his hands still while dissecting mice.  I am always starving about 2 hours before we finish.

2:15ish   After bagels (very little conversation, mostly just furious chewing), i put more media in the spleen and lymph node tubes to keep my cells healthy  (apparently swimming in fetal bovine serum- blood without the cells- makes cells happy), and spun down my liver cells.  I got Katie to help me resuspend the pellets and layer cell separation gradients (kind of like making a black and tan, but with cells and buffer)- these get the crud out and leave me with lymphocytes that i can stain.  While those were spinning for half an hour, i began spinning down the lymph node and spleen cells and resuspending them in preparation for staining.  This takes a while because our two centrifuges only have enough buckets to hold 24 50mL tubes.   The liver gradients were done half way through, and i paused with the spleens and lymph nodes to carefully remove the lymphocyte layers from each gradient  (more eye strain and crick in the neck)  into other tubes to be washed.  Eventually everything got spun down, concentrated, resuspended in staining buffer.

4ish   Next i pipetted cells from each tube into a set of flow cytometer tubes, including a full second set of liver samples for a second staining panel.  Out came the antibodies and the antibody cocktails that i had so cleverly mixed up yesterday.  Each tube got a miniscule volume of antibody mix, which is a brilliant purple color from all of the different fluorophores in there.  Each single stained control got its tiny drop of antibody.  The CFSE (bright green dye that dilutes out as T cells become activated and proliferate) -stained control T cells that i had been culturing since last week came out of the incubator and went into a tube.  (The CFSE stain currently looks fantastic on the flow cytometer, i might add- a first for me.)  Once mixed, I put all of the flow tubes into the fridge for 20 minutes, ran into Isaac who made sure i was doing everything right- i wasn’t- corrected a few of the single stain controls, and then cleaned up my bench and the cart a bit.  And called the program manager back around 5 to tell her that i am unable to TA this fall quarter, no matter how behind on my grad school progress checkpoint list i may be… being a late bloomer  doesn’t bother me (here’s hoping i bloom!).  Also got a phone call from Nicky saying she and her teammates would not be staying at our house tonight… too bad, but now i don’t have to worry about the unsettling number of flies that have migrated from the chicken poop farm outside into the spare bedroom in the basement.

11ish Gah… my eyeballs hurt.  So close to being half way done with flow….!

7ish  Post-incubation in the fridge, i added more buffer to the cells and spun them down to wash excess antibody away,  pipetted off the extra liquid, forgot to resuspend the pellets in fixing buffer, and found Isaac to help me set up his amazing new 8 color T cell staining panel and 4 color B cell, liver antigen presenting cell panel on the flow cytometer.  Awesome.  Awesome possum, you might even say.

9ish  Phone call from David saying that Joakim and the visiting Swede Stefan are leaving the car parked near lab so that i can drive home.  Those boys are just so, so sweet.

11:30ish  Running samples.  After these next two i will stop to fill up the buffer tank and empty the waste tank, and eat some chocolate and go to the bathroom.

I may have just passed from the silly late night lab work mood into angry late night lab work mood.

rrrrrrrRRRRROOOOAAAAARRRRRRR!!!!  Need. Snack. Now.

12am Feeling better after hot chocolate and half bagel.  Contact lenses very blurry.

D’oh!  Failed to collect and stain enough cells from the lymph nodes to get good data.  Pause flow cytometer, stain all of the remaining cells at bottom of lymph node tubes.  Ah- this is how these experiments get so long…

1:45am  Alright!  Running the last 18 samples- new and improved lymph node lymphocytes!  Watch flow tubes carefully to catch when they get sucked dry by the machine.  Put next sample on. So close!

This is dumb.  Next time i will collect cells and stain them, then fix them in one long day, then run them on the flow cytometer the next day- still after hours so i don’t hog the machine, but after a night’s rest.

2:45am  hay-zoos christie.  I’m about to bash this machine in with a baseball bat.  It’s being so finicky and i only have 5 samples left!  5 samples!!!!!

3am   YESSSSS!  Last one!  VEEKTORY!

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Often when we go on vacation for more than a couple days, the basement of the house is relatively quiet and the rats that live in the neighborhood feel they can infiltrate through the cat door and explore.  Then we come back, and Tilly immediately catches one, carrying it around the house like a prize, fighting off the dog for it.  This most recent rat was only half dead- still breathing and aware, but unable to move except for the occasional outburst of squeaking in pain.  While we do like Tilly to catch rats to keep them from living in our walls, we like when she kills them quickly.  This was not one of those instances, and animals in pain cause both David and i much distress.  So i decided that since i have learned through my lab work how to kill mice, i would employ those skills to help this rat pass quickly.  What an odd feeling it was to have decided that.

In lab, when a mouse experiment is done or when it comes time to collect immune cells or blood or the liver from a mouse to gather data, we generally euthanize the mice by CO2 inhalation in a small chamber.  Though it’s apparently the nicest, least painful way for the mice to die- they essentially pass out and then die in their sleep- it’s not enjoyable to watch as they first scrabble around the cage to find air and then lay heaving as they take their last few breaths.   Then, the international animal care committee has a rule that we must kill them a second way to make sure they are dead and do not come back to life only to find themselves in a plastic bag in the freezer- a fate surely more terrifying than dying in one’s sleep.  The second method is cervical dislocation, in which you hold the mouse’s head/neck down between pinched thumb and forefinger, then yank the base of the tail away with the other hand until you feel the skull pop away from the spine. This is painless for the mouse, because it’s already dead.

So, i wrastled the rat away from Tilly and told David to stay inside while i took the rat out back.  David is even more sensitive than me about animals dying, and since i already have my issues and hundreds of dead mice under my belt, i wanted to spare him the experience.  I grabbed the rat with a plastic bag, held the neck, and pulled on the tail.  I’m not sure why it surprised me that a live rat would struggle, unlike the dead mice.  It took me a couple tries to pull hard enough against the rat’s contracting muscles, but once i got it, the body went limp instantly.

I did feel like i had accomplished something, for the rat’s sake and for my, uh, gutsy-ness, but i’m not sure i would do the same thing again.  There’s a sharp difference between seeing an animal die and doing it with your own hands, and i don’t know that most people (including me) are strong enough to have the benefits of putting an animal out of its misery outweigh the toll that killing the animal takes on one’s heart.

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I’m having a mid-PhD crisis.  All of a sudden, i want to be a farmer.  Instead of immunology, i want to study sustainable agriculture.  I’d rather be playing with dirt than with test tubes.  Should be discovering the mechanisms of T cell priming in the liver, instead i’m working out the mechanisms of composting chicken manure.  Instead of immunizing mice against malaria, i’d like to be inoculating my clover cover crop with mycorrhizal bacteria.  Rather be harvesting runner beans than lymphocytes.  Forget flow cytometry- what about nitrogen cycles?  Publish an article in Nature?  Nah, i’ll just share swiss chard and fresh eggs with my neighbors.

Why?

A yearning for the simple life?  I want to fill my days with tending the garden, walking the dog, cooking meals for family and friends, and watching the chickens from a lawn chair.  I want a cozy house on a sizable chunk of land with good southern exposure.  I want miniature dairy goats and a donkey named Guillome who will let the dog ride around on him. Essentially, i want to be the type of farmer who gets to sit in the sun munching on peas and admiring her garden more often than she worries about making a profit.  But i doubt that type of farmer exists- being a farmer is surely harder than graduate school or a career in biomedical research.   So perhaps i’m just lazy and am suffering from grass is greener on the other side syndrome.

A reaction against scientific experiments on animals?  I am an animal lover through and through- always have been- so why did i think i could complete a PhD project entirely built upon mouse experiments?  Did my passion for saving children in Africa from malaria infection blind me to the fact that i would have to kill a shit-ton of mice during 5 years of grad school?  I’m not ethically opposed to scientific research using animals- many important medical discoveries have come from animal studies.  For many diseases, they are the only way forward in the search for vaccines and therapies.  And though i believe in the importance of my research (finding the mechanisms of protective immunity against liver stage malaria infection in order to inform the design of an effective anti-malaria vaccine), and think it merits using animals more than, say, testing cosmetics or diet pills, it’s getting harder and harder to kill those mice.   My project is the envy of many of my colleagues, and is very academically interesting to me… but i’m losing motivation.  And perhaps this pushes my mind to wander toward greener pastures speckled with free-range chickens, frolicking goats, and rescued doggies.

Or just a change in interests?  It’s hard not to become fascinated by something when you really take a look at it.  And it’s hard not to become engaged and passionate about any issue facing the planet when you really start reading about it.  I know i’m putting myself at risk of sounding like an academic activist d-bag, but it seems like i can’t not become interested and passionate about any subject that crosses my field of vision.  I’ve got nerdy, bleeding-heart liberal ADD.  In elementary school i wanted to become a veterinarian (that shoulda told me i wasn’t cut out for mouse experiments) and since then, my interests have ranged from math and physics to American ethnic studies and civil rights, from infectious disease research to global health to international development, from women’s rights and education to economics to the environmental effects of agriculture.   I wouldn’t mind just being an “armchair expert” in these varied disciplines, except that i get so damned carried away and emotionally invested in every one of them, and each topic is more important and urgent than the last.  At first it was breaking the disease-poverty trap in sub-Saharan Africa, then it was investing in women’s education to spur international development, now it’s learning everything about organic and biodynamic agriculture and getting involved in outreach to help small organic farmers get a foothold and help industrial farmers make the transition to sustainable growing methods.  Each new focus is the key to saving the world… if i could only study it, become an expert in it, teach others, influence policy…

But then, that sounds like a lot of work…  when would i have time to garden?

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