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Posts Tagged ‘slacking off’

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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