Static On Film

Have you ever gotten strange artifacts on your films that look like this:

This is the result of static electricity and if you have acclimatized yourself for long enough in the darkroom; you can even see when the static electricity sparks go off on the film.

These artifacts are a pain because they can ruin a perfectly good experiment. For something like western blotting, getting an artifact like the above that covers up your result is annoying but most of the time, you can make a few repeat exposures before the ECL substrate gives out on your membrane. However, if you are working with something that takes weeks to months to develop on film (e.g. S³⁵ in situ hybridization), getting an artifact that covers up some of your most important image results can be very devastating as the entire experiment will probably need to be repeated.

I have had the unfortunate experience of having these artifacts crop up in my experiments. Over time, I came to realize that the main problem lied with the lab’s air conditioning.

Static electricity is a build up of electrons (negative charge) within or on the surface of an object that is retained until it can be released by an electric current or electrical discharge. Water is a conductor of electricity, so in a humid environment, the moisture in the air absorbs and can evenly distributes excess charges, thereby allowing excess charge to leave objects.

My Experience

During my time working in a lab housed in an old building, I have never once gotten an artifact like the above on any of my films (and I had done a lot of film work). Fast forward to my new lab, which was brand new, the artifacts appeared almost all the time I did any film work. My technique, equipment, and reagents were pretty much the same so the only real difference was the lab itself. I ultimately concluded that the problems came from the level or moisture in the air. In my old lab, there was no air conditioning or windows that actually opened. As such, the lab was always humid. In contrast, my new lab was fully air conditioned and the air was always very dry. I tested my theory by having the air conditioning turned off in the new darkroom; while it did get pretty hot and humid in there, importantly, the artifacts stopped.

Suggestions

If you are having problems with artifact on your films cause by static electricity:

* You can try using films containing an anti-static layer, although I still found the artifacts cropping up on them.

* Try preventing the static build up beforehand. If you are using transparency film to sandwich your western blotting membranes, leave them in a humid area. The same goes for the autoradiography film or hyperfilms.

* If possible, switch off the air conditioning (at least for the darkroom).

* If possible, use a humidifier.

Ways To Ensure Experimental Consistency #2

The following is pretty obvious but does deserve a mention because sometimes, the obvious get overlooked.

Aliquoting

Some things don’t handle temperature changes too well and there are going to be times when enzymes, antibodies, etc get forgotten and left out on the bench for way longer than it should. As such, it is good practice to make aliquots because if something goes wrong, you will have backups! For instance, if you want to preserve your PCR enzyme and keep it at maximal efficiency for the life of the kit (i.e. however long it takes you to use it up), make aliquots of the polymerase. That way, the enzyme will undergo less freeze-thaw cycles and if you happen to forget about it and leave it out on the bench overnight, you can just grab a new aliquot. The same applies for antibodies. 

Mixing

Reagents, samples and solutions, etc sit around frozen, chilled or at room temperature waiting for us to use them. While they sit and wait, some chemicals will separate or you might get precipitates (think SDS falling out of solution when it gets cold). If you have solutions in large 1L clear bottles, it would be pretty easy to see, but if you had frosted 1.5-2ml microfuge/screw cap tubes or those brown opaque ones, it’s not going to be obvious (if at all). For instance, if you defrost your dNTP mix for PCR without giving it a good mix before taking a few mircoliters for your mastermix, would you expect to have a equivalent concentrations of all 4 dNTPs? So unless there is good reason not to mix something, its good practice to give small tubes a quick vortex and pulse spin to mix up the contents.

Other Things To Consider

* Use filter pipette tips. These come sterilized and DNase/RNase-free. The filter also prevents liquids and vapors from volatile solutions from going into the pipette barrel.

* Calibrate your pipettes regularly.

* Clean out the pipette barrel. It is amazing how much build-up you can get in the pipette barrel. You probably don’t need to do this as often if you are using filter pipette tips, but if you are not using filter tips, clean it regularly.

* If you make your own solutions, make sure that the date the solution is made, storage temperature and maker are clearly labeled.

* Some antibodies are very temperamental. If you get a batch that works, make sure to order a few more from the same batch.

Rights To Your Research

Who Owns Your Research?

If you are an employee, student, or volunteer in a research lab, the ownership to the research project and the work that you do for it (e.g. experiments, manuscripts, data, etc) belong to your employer. But, that does not mean that you, as the author, have no rights to the work that you have created.

Moral Rights

Some of you may have heard of moral rights but for those who haven’t, moral rights are rights related to copyright. They are rights that are independent of an author's economic rights and cannot be purchased, transferred or waived as compared to copyright. The duration of an author’s moral rights can persist even after the author’s death, although in some countries, the duration can cease when the author dies or cease when copyright of the work ceases. So essentially, even if you eventually leave your lab, you still retain your moral rights as the author of the work.

Generally, moral rights enable an author to claim their right of attribution to authorship, to object to certain modifications of their work as well as other derogatory actions; and to provide a means of redress where infringement has been found. In short, moral rights protect an authors right of attribution, right not to have authorship falsely attributed and right of integrity of authorship.

As a researcher, your written works (such as manuscript drafts, reports and publications) and original experimental designs (including figures and diagrams) should be protected by moral rights.

Infringement Of Moral Rights

Examples of some infringing actions include:

* Falsely attributing authorship to a person who had no part in the writing. This includes adding a senior member of the lab to a manuscript for merely reading a draft of it.

* Attributing work to an author where the work has been modified without their consent and where the modification amounts to a distortion of the original work or it was not reasonable to make the modification.

* Attributing authorship in a way that is not reasonably and sufficiently prominent.

Consent

While moral rights cannot be transferred or waived, an author can consent to breach of their moral rights. This is usually done by written agreement between the parties. Some employers include such clauses in employment contracts so before signing employment contracts (or any agreement), do make sure to go over the documents carefully to know what you are agreeing to. If required, seek legal advice.

Remedies

If infringement has been found, a court may order relief including compensation in the form of damages, injunctions, declarations, removal/reversal of derogatory treatment, etc.

Useful References

* Article 6bis of the Berne Convention

* WIPO Performances and Phonograms Treaty 1996

* Copyright Act 1968 (Cth)

* Copyright Act (R.S.C., 1985, c. C-42)

* Copyright, Designs and Patents Act 1988 (C. 48)

Cell Freezing Protocol

To continue the cell culture theme of the previous two posts^1,2, I thought I would share my protocol for freezing down cell culture stocks.

Lets assume that I have HeLa cells.

Defrosting Cells

If you receive a cryovial of cells shipped in dry ice, either store the vial of cells in liquid nitrogen or defrost the contents. To defrost, immerse the entire vial in a 37 degree waterbath. When you see that the contents of the vial has defrosted to a partial liquid state but still has a frozen block inside, spray and wipe down the vial with 70% ethanol, open the vial in a laminar flow hood and empty the contents into a sterile tube containing warm (heated to 37 degrees) cell culture media. Spin down to pellet the cells. Discard the supernatant and resuspend the cells in 5ml of fresh cell culture media. Transfer the contents into a T25 flask for culturing and expansion.

 

Cell Expansion

With a T25 flask of cells, grow it to full confluence and transfer (by trypsinising or whatever protocol you use to detach/passage your cells) all the cells to a T75 flask. From here, grow the T75 flask to full confluence and split the cells into two T175 flasks. Again, grow the cells to confluence and expand them into a further two T175 flasks. When the cells in all 4 flasks are at full confluence and ready for passaging, use your standard protocol for cell detachment, but take an aliquot to count on a haemocytometer. Centrifuge the tube to pellet the cells as you carry out the cell counting. Calculate how many cells you have in your cell pellet. You will need ~1x10⁷ cells/ml for the Master cell stocks, thus for 2-3 vials, you will need at least 3.5x10⁷ cells (the extra cells you will need for further culturing and expansion).

Freezing

Prepare your freezing media. I would recommend using freezing media made up of 90% FBS + 10% DMSO for the Master and Submaster cell stocks; and 10% DMSO + cell culture media containing 10% FBS for the Working cell stocks. Make sure your DMSO is cell culture grade and sterile. Your FBS should be sterile. If you are using a new bottle of FBS, it should be sterile but if there is any uncertainty just filter it with a 0.2um pore filter.

Have your sterile cryovials ready and labeled. After your cells have pelleted, in a laminar flow hood, pour off the supernatant and resuspend your cell pellet in 5mls of freezing media. Aliquot 1ml per cryovial, cap your vials, and place them into a freezing container (e.g. Mr. Frosty or a DIY chamber). Store the freezing container of cells in -80 degrees for 24 hours.

With the cells that are left over, dilute them in serum-free media and spin down. Pour off the supernatant to remove the freezing media and resuspend the pellet. Transfer everything into one T175 flask. Repeat the expansion process to freeze down your Submaster cell stocks (@ 5x10⁶ cells/ml; ~8 vials) and then again for your Working cellstocks (@ 1x10⁶ cells/ml; as many vials as you need).

After freezing the cells in the freezing container at -80 degrees for 24 hours, transfer the cells to liquid nitrogen for long-term storage. If you need to transport the freezing container from one location to another, use dry ice to keep the contents frozen.

Note

There may be times when a collaborator will give you a T25 flask of cells with media filling up the entire flask. If this is the case, spray and wipe down the flask and put it in the incubator for a couple of hours so that the cells can settle and acclimatize (cells tend to shrivel when cooled).

In a laminar flow hood, open up the flask and pipette out the excess media and filter it into a sterile tube or bottle for later use. Leave enough media in the flask for the cells to continue to grow.