Cells

You know those little guys that we all learned in high school biology that are the building blocks of life? Yea, they're important. I do a lot of work with cells, and I want to talk about some of those procedures.

To begin with, the type of cells that I work with are Human Embryonic Kidney 293T cells. Known as HEKs or 293Ts. A cell line is a group of immortalised cells. You can order them from a company, and you know what you are getting. HEK 293s were originally from a "healthy aborted fetus" in the 1970s. The number 293 comes from Frank Graham (the guy who immortalised this cell line), who numbered each of his experiments. Thus, HEK 293s came from his 293rd experiment.

HEK 293s are not just plain human cells. A bit of adenovirus 5 DNA was added and incorporated into the human chromosome. This is what gives them their immortality.

Ours are not just plain 293s, but 293Ts. This means that they are able to be transfected with plasmids easily. More on that later.

The way we work with these cells is very cyclical, so I really could start anywhere in describing the process.

We'll start with a flask filled with cells. The cells are in media (read food). We use DMEM as our media. DMEM is composed of amino acids, salts, glucose, vitamins, iron, and phenol red. Phenol red is very important. It is a dye that changes color based on how acidic it is. Mixed into our DMEM is fetal bovine serum (FBS) and a penicillin/streptomycin mixture. The FBS is what is left of the liquid portion of fetal cow blood after it is allowed to clot. It does not have blood cells in it, is low in antibodies, and has a good amount of growth factors. The pen/strep mixture is antibiotics so that the cells don't get infected.

You pull your flask out of the incubator (37 degrees C and 5% CO2), and you notice that the liquid (media) is a dull orange. Time to change the media! Why? Well, remember that phenol red? It starts out a reddish-pinkish color. When it gets too acidic (cell excrement makes it that way), the cells will die. Imagine living in your own poop. Even if you get nutrients from the air, after a while there is just too much poop, and you suffocate from it. Same sort of thing. The change in color signifies that it is becoming too acidic. These particular cells are called adherents. This means that they stick to a surface. In this case, they stick to a side of the flask (whichever end was down). So you flip the flask over, and all the liquid is on the opposite side from the cells. Now you can use an aspirating pipette (also known as a vacuum) to pull out the spent media. If you just want your cells to grow more, you will add more media. This is known as changing, swapping, or replacing the media.

Let's say that before you swapped the media, you looked at your flask under a microscope. You found that there was some overcrowding going on. This is another leading cause of cell death. If cells do not have space to grow in, they die. Sometimes something peculiar happens and the cells on the edge will start growing up the walls. Sometimes, they don't have good anti-gravity skills and the off-the-wallers become curlers. A film of cells will curl up off the wall and back into the cells. It may look cool, but it definitely means your cells are unhealthy (I know from experience).

How do you deal with cell-overcrowding? Decimation. You suck off the old media, and put in a little bit of trypsin-EDTA. The trypsin will cleave proteins (it's actually one of the digestive enzymes that digests proteins in humans), and the EDTA prevents the cells from clumping. You leave that in the incubator for five minutes, and all your cells will come off the walls/floor of the flask. You then spray them down with media so any clingers are forced into the solution. You pipette up and down to make sure everything is mixed nicely, and usually you pull off 9/10 of the liquid (and thus the cells) in the pipette. Then you vaccuum them. You are left with 1/10 your original cell count (-ish) and you add more media to get back to your original volume. I have found that doing this generally gives you three or four days before your cells are confluent (overcrowding) again.

We have cells. We know how to maintain them. What's the point? In our case, the point is usually transfection. Transfection is introducing foreign DNA for the cell to incorporate in its own genome. You put your cells in solution, and instead of vaccuuming a large portion of them, you then add that portion to a different flask. You then add an appropriate amount of media, and do some heavy mixing. Then you add about 10 ml of this new solution to your cell culture plates. You may know them as petri dishes. Your cells will grow and flourish (hopefully), and reach confluency.

After a couple of days, when it is time to change the media, you can transfect them. You put a bit of the DNA that you hope to express in a mix with media and a special reagent that will make a hole in the cell membrane for the DNA to go through. You add a few drops of this mix to each plate, and the new foreign DNA gets pulled into the cell. If everything worked well, the cell will think, 'Huh! There is DNA outside of the nucleus. Better put that back where it belongs!' The cell adds the new DNA to its own, and then acts as though nothing is different. In our case, the foreign DNA will code for a particular protein.

After another couple of days, the cells had enough time to produce that protein, and you can harvest them. This is done by basically scraping each of the plates, and sucking up the cells that were on there. There are a number of things that can be done after you have harvested the cells, but that is outside the scope of this post.

Now, you are an expert on cell culture! (-ish)

General Updates

A lot has happened in the last few weeks. To start with, I no longer live with JK. He went away on vacation for about a week, I watched Hershey the dog, and when he came back, I moved out. This was planned, and I gave plenty of headway.

I currently am living in an apartment that's actually in the center of town. It is a little bit less than two miles to get to the base. In fact, I turn right out of my apartment, turn right at the end of the street, and if I keep going straight, I end up on base. Win.

AK finished up school for the year, and came down to live with me. Timing worked out really well there. She finished up her finals and whatnot before the end-end, and I had a meeting to finish up Division II with JM and CJ. I went up to Amherst for my meeting, brought some great beer to celebrate (Three Philosophers. If you haven't tried it, do so). JM and CJ asked me all sorts of questions to gauge the sorts of things I have learned in the last two years. This is, of course, after I had given them a portfolio and retrospective essay. That way, they knew about which things to ask questions.

After the meeting, AK and I drove down to my folks' house. We had some dinner and grabbed a few things that I had not brought down the first time around. In the morning, we drove down to Frederick. And, I, smartypants that I am, went in to work. I had a nuclear extraction to do (I will explain that in more detail in a different post), and it took me about five hours. I had just driven about five hours, and then I worked for five hours. A glutton for punishment.

AK and I went tag saling last weekend. We bought a table, chairs, a microwave, a toaster, and various knick-knacks. Our apartment is starting to look homely.

AK is on the hunt for the elusive "job." She has applied to probably 30 places by now... So far, no dice. But, not all of them have rejected her yet either. If nothing comes, she is planning on volunteering. Probably with the local animal shelter.

I have been falling more and more into a routine. Aside from nuclear extractions that take about five continuous hours, it is mostly hurry up and wait. Right now, I am waiting for a gel electrophoresis (1.5 hours). After that, I do about ten minutes of stuff, and wait another 1.5 hours, etc.

The smallest things

Today, a package came for AB (not an altogether uncommon experience). It was probably about 8x6x5 inches. Inside it was another box, about half the size, with nothing else. Fischer Scientific (the sender) is claiming to go green, but I have yet to see any evidence.

Inside this second box, was... labelling tape. Twelve rolls. This is basically colored masking tape, but a little bit stronger so you can color code/label your experimental items. There was only one roll each of blue and orange, but two rolls of white, yellow, green, pink, and red. I asked AB, and he said that blue and orange are the most popular colors so they sell those separately. No, that does not make any sense to me either.

AB decided he had to put the colors in the right, most useful order. We have a tape dispenser that can accomodate eight rolls of tape, so that is one of each color, plus one of scotch-type transparent tape. AB spent the next five to ten minutes putting them in the "right" order. Previously, I had just used whatever tape was closest or whatever, but now it seemed like there was a correct and incorrect tape to use for different circumstances. I asked AB. He said, "Of course the colors have meaning. Everything has meaning." I made a face that begged for a more complete response, but none was forthcoming.

He had seven rolls on there when he said, "Hmm, but where does the pink go?" He thought about it for a moment, and took a few rolls off, put the pink on then replaced the rest. The final order is transparent, orange, red, pink, yellow, green, blue, white. He glanced up at me and looked really pleased with himself. I had an urge to pat him on the head and say, "Good boy!"

It's the smallest things that make people happy.

Umm...News?

First, I realize it's been a while since last I updated you on life in the lab.  I have been falling into a bit of routine.  I now have my very own laboratory notebook, in which I need to detail my experiments.  It's like I'm a real scientist.  I have also gotten pretty good at all aspects of PCR.  Yesterday, I loaded a gel after PCR, and it was a real beauty!  Even better, it showed that everything worked very nicely.

Hampshire's only molecular/cell biology class with lab is called Gene Cloning.  It is every January.  It is a three week, 8 hours a day, intense lab boot camp.  My thoughts: how could they possibly learn anything useful (read: do any experiments well) in such a short period!?  I have been at this now for 7 weeks, and I work eight hour days!  I am only starting to get good at some things.

Today and tomorrow are the Spring Research Festival.  I went and explored.  It was awesome (in the quite literal sense- I was filled with awe).  Think huge circus tent.  Or exhibit hall if that suits you.  Eight rows of tables.  Easily 50 tables long.  It was huge.  If you register, you get a free t-shirt upon entering.  It's a nice one too.  I thought the easiest way to approach this was to start on the right and work my way down the tent.  It seems that this is how it was organized, so it was a good choice.  

Most of the tables on the right were Department of Defense, National Cancer Institute, Fort Detrick related things.  This included this included childcare services, Fort Detrick's recycling program, NCI-F's library services, etc.  I got a very nice pen from USAMRIID.  I think that is the Army's bio-warfare science unit.  It is a very nice pen though.

The next section was posters.  This was mostly postdocs showing off their work.  I felt bad for them.  I walked down one row of posters, and it was enough.  I emerged on to something that my mind had a hard time coping with.

I've only been to one kind of trade show in the past.  Jewish.  I have been to lots of Jewish conferences, and they often have exhibit halls.  Publishers show off books, leatherers show off book-bindings, there are pretty things wrought in silver, and other pretty things woven of fabrics.  A science trade show is like walking onto a movie-set for some futuristic something-or-another.  There was a booth filled with really pretty tools for cutting, namely scalpels, scissors of various shapes and sizes, and tweezer-like objects.  There were incubators from cute to discreet to "huh, that looks like an incubator," with sales-people showing off all their interesting features.  There was a robotic pipetting booth.  Basically, you use a computer program to tell it origin and destination information (and it is all color-coded and click friendly), and the robotic arm pipettes just the right amounts of the right stuff into the correct destination.

Food was brought by ZiPani, a sandwich place in town.  I went there once with AK because a name like that might lead you (as it did us) to believe that it is a bread place.  I got a sandwich at the ZiPani stand (it was pretty cheap and there was no sales tax!), and sat down out side at one of the picnic tables.

One of the exhibitors joined me, and his business is based in Natick MA, so he was quite familiar with Amherst, and even knew about Hampshire.  He was a real pleasure to chat with.  It was fun.  Aside from the requisite business name and his own name, his name tag said, "Likes broccoli and is good with kids!"  He was a pretty cool guy.

I came back and had to do a nuclear extraction.

A few days ago, I put some cells on cell culture plates, and transfected these cells with some DNA.  This means that I inserted DNA into the cell, with the hopes that the cell would then express (manufacture) the protein that the DNA codes for.

After the cells have been expressing this protein for a couple days, I basically scrape the cells off of the plates, and put them in test tubes.  The cells then get put in a centrifuge, and I separate off the liquid.  I then add materials that will allow me to extract the nuclei of the cells after spinning them down again.  

Today, it did not work.  I noticed when scraping the cells that they were "gummy."  As in, they stuck together in long strands.  When I sucked them up in my pipette, they pulled like boogers.  I put them in the centrifuge, and when I took them out there was no pellet.  Normally when you run the centrifuge, all the heavy stuff sticks together and forms a "pellet" at the bottom of the tube.  You can then suck off the liquid, and you are left with the pellet.  Either you add something else at this point and re-centrifuge or you stick it in the freezer depending on what you are trying to get from the pellet.

No pellet formed.  We thought that maybe I didn't spin the centrifuge fast enough, and thus there was not enough force to separate the pellet from the liquid.  We spun it faster.  No good.  A few of the tubes looked like they might have a pellet, but when I tried to pull the liquid, the boogers came with it.  

So what does this mean?  It means that my cells died before I scraped them.  I admit that I did not check on them under a microscope since I transfected them (two days ago).  But they should have been healthy.  We were using a different serum this time.  Normally we put FBS (fetal bovine serum) in the media, and the cells grow very happily.  However, we were out of FBS, and we used a different company's FCS (fetal calf serum).  These really should be the same thing.  So naming conventions aside, the difference was that the FCS was heat-treated.  The going theory at the moment is that my cells were acclimated to our regular serum/media combo, and the shock of changing this was detrimental to them.  Who knows?  No use speculating.

So on Friday, I will start a new transfection process.

In other news, NIH has apparently censored signing in to a blogger blog.  I am able to read blogs, but theoretically not post or comment.  I happened to have set up a way to blog by email.  Unfortunately, I can't check formatting and whatnot.  Hopefully that works.

--
-Tal