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So, I’ve been trying to pin down a hard-to-define phenotype in some cells this past few days. When I knock down a protein with shRNA virus, I get a qualitative phenotype that I’m trying to make into something quantifiable. I’d spent the past couple of days going back and forth, trying to figure out what’s wrong with the knockdown cells. Today, I finally decided I’d make a big montage image so that I could see a large number of control cells right next to a large number of knockdown cells. I spent a long time taking pictures of 50 cells from each sample, ready to compile into a big figure. I save all the images from the microscope computer, import them onto my own notebook… and then realize that the damn microscope software defaults to saving them as 8-bit images instead of 16-bit images. So every image is a useless basically blank image, the software having thrown away the data hidden in the “blown out” highlights that can only be restored from the 16-bit part. And there’s no automatic saving of a master file, as there is on the other scope I routinely use. So, now I need to repeat the experiment. I guess I’ll know for next time.

Thankfully, I received a text message just as I was realizing my mistake. Four friends were going to Brasa in my neighborhood for dinner in celebration of one of them winning the Green Card lottery. I rode my bike over from campus and had an awesome dinner – they’re definitely a group of friends I’ll really miss when I leave here.

The university’s graduation ceremony for graduate students was today. Three of the students in the lab are due to be done over the next few months, including me: the other two walked, but I decided not to. The ceremony didn’t seem like a big deal to me – my family will probably come over for my thesis defense instead, whenever that is. It was good to see all my friends in their gowns with their families. I met them after the event at the bar (but no drinking for me), and we all hung out and talked for a while. I’ve made good friends in grad school, for the first time being around a group of people who were mostly interested in the same stuff I am. Everyone’s moving away, though, on to postdocs or jobs, which is pretty damn sad.

I’m not sure what I’m doing yet – I’ll probably be graduating within the next 4-5 months and perhaps staying on in the same lab for a while, or moving back to Europe, or staying somewhere in the US. So I have no idea, really. Paris / London / Amsterdam / Switzerland / Boston / San Francisco, maybe? It’s a bit uncomfortable not knowing. It’s uncomfortable to know that this major change is coming soon and I can’t make a start on looking seriously for jobs until I have the publication I’m working on submitted to a journal; it feels like I’m leaving it much too late, but I’m told there’s no point in even approaching prospective mentors until that’s done. I guess that makes sense. But it’s also exciting – I can do whatever I want with my life. No kids, no significant other, no strong emotional or practical pull to a small area (although I’d like to be nearer my parents).

Anyway, the graduation parties are continuing as a house party. I’m heading out.

So, those chromosome spreads were apparently roughly what I was supposed to expect, according to my advisor. The fully-exploded chromosomes are apparently usually only obtained when a different method is used. This is OK, then.

Meanwhile, I was also trying to see the distribution of some chromosome proteins by paraformaldehyde fixation and DAPI counterstaining. The fixation went exceptionally well (mitotic chromosomes did not swell or become indistinct as they sometimes do in methanol), but visualizing the mCherry had a couple of problems. The signal was fairly well preserved despite the paraformaldehyde, but was difficult to see against heavy background from something in the preparation (which by this point was mostly just cells and Vectashield with a little left over PBS). After setting up a series of test slides (PBS alone, water alone, Vectashield/DAPI alone) I discovered that the Vectashield with DAPI was the source of the background. Next time, I may have to DAPI stain and wash my cells separately from the mounting process (and use Entellan for the mounting instead of the Vectashield). But this is progress.

Image source: http://biology.ucsd.edu/classes/bimm110.SP07/ lectures_WEB/L08.05_Cytogenetics.htm

I’ve been trying to characterize some cells to work out how they’re growing under certain conditions. To do this, I’ve been trying chromosome spreading and Giemsa staining today. I’m working with HeLa cells, and there’s quite a bit of skill involved in getting it right. Basically, it involves getting some cells grown in culture in a dish, harvesting them, making them swell up in water and then rapidly fixing the chromosomes in a mixture of acetic acid and methanol. You take these swollen cells and drop them from a short height (a couple of inches) onto glass slides, let the slide dry and then stain them with Giemsa stain to make the chromosomes purple. Continue reading →

Today, I’m still recovering from the weekend. I don’t like alcohol; it definitely does something weird to my head that lasts for a few days after a night of heavy drinking. I think I’ll have to give it up.

In lab, I spent the morning checking some TOPOIIA fusion protein clones. After getting to the end of the digests and seeing that they didn’t look anything like they should, I shared my confusion with my advisor, who was also baffled. It wasn’t until I wrote up the results and put the gel images in my notebook that I realized I’d digested the samples with the wrong enzymes. I wouldn’t have realized this without keeping it all together in my notebook, which I guess goes to show how helpful being organized really is.

I have a lot to do to finish my work for the localization paper. Knockdown/colony formation assays are turning out to be slightly ambiguous (although not too bad – this is probably largely paranoia). I’m working on doing chromosome spreads instead, to figure out the mitotic index of some cells. Just thinking about this reminds me that I resist doing assays that require skill to interpret, like this one. And that I haven’t done much data analysis. This is something to work on.

At the student union today, some girls asked me to stop and answer a question on video for their biology class (“only, like, twenty people will see it!”). I hesitated, but agreed. “What is the only form of birth control that’s 100% effective?”. Shit. This felt like a trap. Regretably, I answered.
“Oh. Really?” [long pause, sigh], “Well, in theory it’s abstinence”. They say that yes, it was a trick question and I’m congratulated. Camera off, they’re done. Fuck. I ask them if it was really for their biology class, and they confirmed it again. I hope it really was just for that, and words haven’t been put in my mouth for some churchy purpose; I’m not even convinced my answer is really any more correct than any others I could have given. Should have suggested Saddlebacking.