The Half Decent Pharmaceutical Chemistry Blog

No, nothing to do with women’s football in Iran. I am talking about sonication. My supervisor is almost fearless: she is only fears the sonicator. And contaminations. And radioactive labels. And counter Geiger. And ethidium bromide. And the junkies in the park where the lab is, especially when she goes to work in the weekend on her own (but, frankly, who isn’t?). However, despite these marginal fears, she is lionhearted. For what concerns the sonicator, this is a new one, as I hadn’t used it until I began to do immunoprecipitation and, on this occasion, employed ultrasounds when shearing chromatin.

She took me to the basement level of the building where, close to the autoclave, our shiny, new sonicator is. There is obviously a good reason for keeping such an instrument there: the moment you turn it on, it starts making an unbearable noise. I had worked with less powerful models in the past and I can only assume that, as a general rule, the more powerful the machine, the greater the noise.

Still, noise is the least of the problems: apparently, ultrasounds can damage your gums and teeth. More worryingly, she warned me that, as a man, I should make sure NEVER to stay in the same room with the sonicator while it’s on as this is likely to result in permanent infertility in men only.

At first sight she might be right: ultrasounds are very good at destroying spermatozoa, ear-protection are required (and much welcome), ultrasound can disrupt some minor vessels (when using it at analytical chem. lab-courses, to facilitate acetylsalicylic acid dissolution, if I remember, we were told it could result in the destruction of most superficial capillaries), but, for the life of me, I haven’t been able to find anything that backs my supervisor’s fear for the danger linked to a small sonicator working under a sort of removable, thick, plastic cabinet.

Is this fear totally irrational? As far as I can tell, it is. But I cannot entirely blame her as, if I have learnt anything during my first four months in the lab is that your fear is often useful, keeping you alert and concentrated.

I’ve never been the type of person who makes New Year’s resolutions. January 1 is only the first day of a month. Perhaps, August is the moment for your resolutions: you’re on holiday and in between two working periods. In a week I’ll be back in business and fully operative in the lab, although, as I’ve already written, I haven’t taken any break whatsoever despite promises and common sense. Because I worked in the lab on Christmas day, I don’t see I shouldn’t do the same on New Year’s day when, traditionally, whatever you do is likely to be done for the rest of the year. So, blogging and working are to continue for the remaining 365 days (as this is a leap year: hence, election year in the USA and Olympics).

For what concerns the blogging, there is a big news: my old computer is about to get a massive boost as I came across a guy from Florence who sold me two 256 MB SDRAM PC133 cards for less than 40€. Therefore, what is now a sluggish 64 MB infuriating non-sense, is to become a decent 512 MB beast, with positive consequences for the blog, of course. If not for its contents, at least for the stuff garnishing it (pictures, layout, etc.).

As I said, I went to visit my cells on Tuesday. In afternoon, of course, because, like any other normal guy in his twenties, my had been to a party to celebrate with noisy fireworks and lots of champagne the new year. Did I enjoy it? Well, it was better than the previous 6 ones, but what I really liked was me solitary walking back home at 2:00 am (arriving 45 minutes later) from the party, through the entire centre of a city in a sort of after-party, hung over condition, with several people going home happy and drunk, wandering through the hospital buildings, trying to avoid noisy people (successfully, as I know my city very, very well), listening to melancholy songs of the Interpol. That was awesome!

Still, what I did in the lab in afternoon was pretty awesome, too. I was there to find if a certain cell line had survived a certain treatment with aphidicolin and camptothecin, two drugs I am using a lot, at the moment. That took a little bit less than expected and therefore I had some time to dedicate to my flasks. Actually, believe it or not, for the very first time in my, so far short, career, all the flasks demanded to be split and get fresh medium. That was great because I could sort of synchronize every line, bearing in mind their differences.
In fact, although they are all fibroblast, the two mutant lines, predictably, are as fragile as hand cut Bohemian crystal vases, in the sense that the moment they reach 100% confluence, they begin to quickly detach themselves. CS-A lacking ones (I’ll explain this soon), in particular, should be handled like Hannibal Lecter…

Mind you, I think I couldn’t have chosen two better things to do to start the year. Oh, and there is a third thing: last year, this was an awful period, being busy preparing exams, but I none the less managed to post regularly even staying up late at night. It was a good habit, because this has always been the calmest occasion for recollecting my thoughts as everybody sleeps. That’s why I’m posting when it’s 1:30 am here. I'm on holiday, apparently...
A couple of hours ago I stumbled upon a stupid film. A man said: “I’ll sleep when I’ll be dead.” That’s probably a good New Year’s resolution.

I am a big liar. On Christmas, we should all behave better than usual and, perhaps, spend more time with our family. In a nutshell, Christmas is perhaps the only day when people depressed because of their usual hard-working and much disliked job, can, in theory, either do what their really want or dedicate more time to their family. I totally agree with the former and, actually, go one step ahead and ask why we shouldn’t do this on ANY given day. You’ll agree it sounds like a beautiful, naive utopia. By the way, this doesn’t cancel the opening statement: I am a big liar. You see, my supervisor was categorical when told me not to work through Christmas holidays. I should have killed all my remaining cells. If I wanted, I could freeze some from each cell line, so that back to work, I would have had fresh cells, likely to restart growing more quickly.

I did that but, instead of murdering the leftovers, I kept them. And I’m using them in my experiments which, to go straight to the point, haven’t stopped for a single day. The problem is that in January, I’ll almost certainly have to work at two projects: one for my graduation, the other just to help the rest of the group (that is about to lose a vitally important component next month). What’s more, January will be a particularly packed month for me, so, I cannot waste time. However, I’m not working full-time, but use to go to the lab only in the afternoon, when I’d probably annoy myself. Maybe I’m already become an obtuse workaholic, but I’m not helped by the atmosphere at home which is, well, not very homely as always.

Culturing cells is a little bit like gardening. If you like gardening, you’ll agree you don’t stop looking after your plants just because it’s Christmas. Like a gardener, I also look with pride at my flask, especially before leaving the building, staring at the beautiful colours a flask displays when you’ve recently put fresh medium in it. To sum up, I really enjoy it and don’t see why I shouldn’t do what makes me happy in this joyful and laid-back period of the year, just because no one likes what they do as a job. I mean, if I could, I’d definitely culture them at home, where I dream of creating a fully functioning tissue-culture lab one day. In fact, the only drawback is that outside is very cold and it takes me nearly 30 minutes to go there by bike.

It’s widely believed that plants, as many other living beings, receive a positive influence from listening to some types of music. I mean, it’s not a good idea to play heavy metal in a greenhouse, but, maybe, CERTAIN plants will enjoy reggae tunes, won’t they? Classical music, however, is the most popular choice, by a large margin.

The problem with classical music is that, sadly, I don’t like to listen to it while working very much. A radio station is undoubtedly a nice pick: you listen to the news, the latest singles, some programmes are funny, others very informative, etc. But do cells really care about that? I sincerely doubt it. Luckily, I thing I’ve found a brilliant solution, something that increase the feel-good vibe in me and in the cells, something that enhance a sense of well-being, something that softens and smoothens the atmosphere: lounge music.

Yeah, many of you hate that kind of stuff thinking it’s either boring or too posh or not manly enough, but I don’t care: as long as it relaxes me and keep me concentrated, thanks to its particular rhythm, it’s perfect. In particular, I chose a compilation whose title reminds of culturing, while the cover matches perfectly idea of feeding (actually focusing on a pretty fundamental and primeval concept of being fed): Lounge Culture Vol. 2.
To create the perfect habitat, though, I firmly believe I should add candles and/or softly glowing lights, garnishing this scenario with exotic incenses, but this is obviously forbidden in any lab, not to mention places where you sterilely handle cells. Nevertheless, forget about the incense thing: wouldn’t that be a lab where you wouldn’t mind to spend a lot of time?

I must admit, I haven’t seen the film mentioned in the title of the post, but I guess that’s something I share with many of you. We all know what it’s all about: a skinny, red-haired, German, inexpressive girl, who runs for all the damn 81 minutes the film lasts, trying to find the money to save her boyfriend, everything garnished with techno soundtrack. How disgusting is that?! Frankly, only a completely untalented director could have come up with such a stupid idea. However, in the video-clip era, some useless critic might have been crazy about such a pointless thing.

Annoyingly, this film popped into my head this morning, when I was to conclude my daily session in the tissue-culture lab. Perhaps that was a consequence of the appalling headache I was beginning to suffer (and, partly, it’s not completely gone yet). This was, indeed, a nice reward for my committed work: as said last week, I’m to assay cytotoxicity and to get this job done (quite) precisely and quickly, I’m going to employ MTT. So, plainly, I was plating out some cell-lines, as I’m first about to determine the ideal number of cells for each line. This involved preparing lots of different dilutions and some counting with…,well, the counting chamber. Thing is, that lab is nicknamed “The Fireball”: there wouldn’t even be need for an incubator there but you could perfectly leave your flasks on a table and cells would grow normally.
So: a very hot room, a long work and headache. How fair is that?

Still, I finished with approximately 2 millions of cells to be stored in a so-called cryotube to, as you might expect, cryopreserve them. And this brings back the odd image of that hopeless woman running. Let me explain. Once you’ve neatly concentrated your cells in 1.25 mL of cryo-medium (argh!), you have to place the test tube in the -80°C freezer (before eventually storing them in liquid nitrogen) as quickly as possible. Even when you suspend the cells you can’t waste any time: in fact, after a 5 minutes 1,100 rpm centrifugation (at room temperature), which yields a lovely pallet of cells, you discard the supernatant very quickly (it still contains a bit of the trypsin you used to detach them from the flask and, at this point, you want keep all of them as a pallet) and suspend them again in a small amount of cryo-medium before you put everything in a test-tube.

There are different “recipes” for cryo-media, basically function to the type of cells. For today’s primary fibroblast, the chef, err…my supervisor told me to use a pretty standard 50% FCS, 45% DMEM and 5% DMSO (if you don’t know what the first two abbreviations stand for, shame on you). Yes, there’s a bit of chemistry in this post! DMSO is always added and it’s a fundamental ingredient being a cryoprotectant: if not present, cells will inevitably be damaged by the formation of ice crystals.
With the cells in the tube, to do things by the book you should allow them to cool down slowly but constantly, before storing them at -80°C or whatever. There is a variety of tools to complete this task but we don’t have any of them and, as far as I can tell you, although we’ve been recently awarded a mind-blowing 40,000 € fund from a private foundation, as far as I can tell you, we’ re not going to spend a cent on such a device either. So, what we do, instead, is placing the tube in the fridge the Fireball is featured with. When it’s cold enough, you put the cryotube in the freezer, which is in a separated building: so, it’s not unusual to have, say, a run in the park between the two buildings, generally wearing a shirt because the lab coat must remain there to avoid contaminations and you’re not in the mood of wearing anything more because you are very hot. However, you have to be fast not to shock the cells with the different temperatures (from +4°C in the fridge, to +38°C in the Fireball, to whatever is outside, to -80°C).

Run, baby. Run.

Two days ago, one of my colleagues asked me if I would have dressed up in a costume for Halloween. If I were an American, which means I’d feel the need to celebrate it in some way, I think I’d dress like HAL 9000, from recently-mentioned “2001: A Space Odyssey”. That’d be a very cheap and simple costume: all you need is a huge, black, cardboard box (I could find plenty of them at the chemist’s where I used to work, although, of course, I’d have to paint them), with a red, glowing spot in front. Then you start to make intelligent remarks, showing off your (intellectual) superiority, with a calm, monotonous voice. Not the best way of making friends at parties, I’ll admit…

However, because I’m Italian, today is just Wednesday, and I usually don’t wear costumes on Wednesday. Still, yesterday I told my supervisor I was going to the student office of my faculty to hand in a stupid certificate, and she said I had to be quick because “at 11 o’clock, we’ve to visit Victor2.” Suddenly, the image of HAL 9000 popped into my head. Were we going to meet an intelligent machine, which we had to convince it was reasonable and logic for us to use it? Is Victor 2, the stepbrother of HAL? Would Victor2 eventually kill me, if I accidentally made a mistake and ruined the assay? These were some of the eerie things I was considering while walking in a cold rain, which reminded me more of Blade Runner rather than 2001.
“Hi, Victor 2”
“Good morning. You’re late: problems with the weather?”
“Yes, a bit.”
“Shall we start? I have a busy day, as you can see from the dairy on the shelf.”
“Right. We have to carry out a cytotoxicity assay and we’ve chosen to use MTT. So, plainly, we’d like to start measuring absorbance as soon as we receive the tetrazolium bromide. Thing is, neither of us has ever used this particular technique, so, we’ll have to work out a standard protocol, before we could really start the experiment.”
“The MTT assay is, indeed, an excellent option: you dissolve a yellow tetrazolium in your medium and then those cells that are still metabolically active reduce it to its purple formazan derivative with mitochondrial dehydrogenases, yielding crystals.
The formazan precipitates and must be dissolved: for this task, you can employ either an acidic, alcoholic solution (generally a 0.1 M HCl in isopropanol) or, as people often do here, 0.01 M HCl in 10% SDS. Then you’ll need me so what’s what: the absorbance should be measured at 570 nm, but my instructor built me with a 595 nm filter. No one here has ever had any problem with that. There is another small issue: theoretically, you should subtract a background absorbance measured at 690 or 650 nm, but, again, my instructor didn’t fit me with a filter that allows you to read at that wavelength. However, there is a smart way of solving this: do not put cells in a well of your plate so that it will serve as reagent blank. What kind of plate do you want to use?”
“A 96-wells one.”
“The average volume of cells per well is 100 μL. To determine the best number of cells for the assay, draw first a calibration curve at least in duplicate and select the value that gives you an absorbance between 0.75 and 1.2.”
“Right. So, I think we’ll meet again next week, to test which concentration of cells is the best for the job in hand, ok?”
“I am putting myself to the fullest possible use, which is all I think that any conscious entity can ever hope to do.”
“Uh…”