Category Archives: FACS

FISEB 2014 – day 2

Due to crappy Wi-Fi at hotel, this entry will be short. I’ll try to expand once I get back home.

Anyway, today was very interesting.

At the “early bird” session, I heard about CyTOF. Essentially, instead of using a few fluorescent markers for FACS sorting of different cell types, they offer conjugating the tagging antibodies with rare heavy metal isotopes. they claim that these are not found in cells, so the background should be zero. They have >30 different isotopes they can use, and the detection is by mass spectrometry – so very accurate and distinct identification.

Next was a session on gene expression. I won’t go into details, particularly since much is unpublished yet, but Tzachi Pilpel’s talk was amazing. Who knew tRNA may have anything to do with cancer research?

As per usual, Orna Amster-Choder talked about RNA localization in bacteria with lovely images and great data.

Jeff Gerst from Weizmann discovered a possible new mechanism of mRNA transport in yeast, using the MS2 system in very neat ways.

The next session, called “oral poster 1”  featured short talks. The most interesting to me were about mRNA methylation and about how the DNA sequence surrounding consensus sequence for DNA binding proteins affects this binding. some nice insights.

The last session I attended was about the effect of tumor microenvironment on tumor progression and treatments. Heard some amazing stories. Hope still exist to cure cancer…

Tomorrow is my lecture. Excitement!

FISEB 2014 meeting -day 1

FISEB meeting happens every three years, and it includes participants from 28 different experimental biology societies in Israel. It is the best meeting to learn about biological-medical research performed in Israel at all fields and doctrines.

4 days, 8-10 parallel sessions, hundreds of lectures, >1000 posters, >2200 participants.

The first day started by a plenary lecture by Aryeh Warshel, Nobel lauret. He is really far from my field, and his lecture was very much confusing to me. But he has nice cartoons 🙂 The bottom line – enzymes are able to catalyze reactions due to electrostatic connections that are maintained stable (unlike in water).

From the afternoon sessions, I chose “signaling pathways & networks”. Relevant to this blog:

Yoav Henis from Tel-Aviv Uni. talked about oligomerization of TGF-beta receptors. he used a method he calls “co-patching”, which is essentially IF with two different antibodies for two receptor subunits. homodimerization will yield single color “patch” whereas heterodimerization will yield an overlap of both colors (co-patch). He then looked at the % of co-patch with different receptor subunits with/without ligand, or with mutants.

Maya Schulinder from Weizmann Institute talked about the contacts between mitochondria and other organelles (ER, vacuole) in yeast. These contacts are important for lipid metabolism. She new about the mito-ER contact but found there must be a second contact (bypass mechanism). She used an interesting screen method to find the bypass mechanism to the mito-ER contact: she expressed one of the contact protein as a GFP fusion. She expected that if the bypass mechanism and the mito-ER contact “share the load” of lipid metabolism, then deletion of the bypass will increase the number of the mito-ER contacts to compensate. Using automation, she imaged 6200 deletion mutants (from the yeast deletion library) each expressing this GFP fusion. As expected, she found 4 candidates which turned out to be very interesting.

Roni Seger from Weizmann showed that targeting the nuclear localization signal of ERK can be a novel cure for certain pathologies, including certain types of cancer.

On the other hand, Maya Zigler from the Hebrew Uni. suggested another new idea to cure cancer – by inducing the surrounding immune cells to destroy the tumor.

Ido Amit from Weizmann as well told us that we may not really know all the different types of cells that exist. What most people do, particularly in immunology, is rely on one or two known “markers” and use FACS or other methods to sort the cells based on these markers. However, some of the markers overlap. and there may be cells for which we do not have any markers and they “disappear” in the crowd of unsorted cells. or, the could be further sub-types we do not know about. So he approached the problem in an unbiased way – he took all the cells in the spleen, and did single cell RNA seq to individual cells from the spleen. Thus, each cell type has several hundred/thousand “markers” based on gene expression profiles. Not only did this method agree with the common FACS sorting markers, but he identified several sub-types unknown before.  Expect his paper this month in Science. His paper just got published in Science.

Finally, Yaron Shav-tal from Bar-Ilan Uni. used the MS2 system to study how perturbing the signaling pathway of serum stimulation affects transcription of beta-actin gene. As per usual – very neat job and interesting results.

Fluorescence Spectrum Viewer

During training on the FACS machine in our faculty facility, I encountered the Fluorescence Spectrum Viewer  from BD bioscience.

It is a JAVA interactive site that allows to to view excitation/emission spectra of up to 8 differnt fluorophores simultaneousely.

It lets the user to choose the desired excitation laser, and shows the excitation & emission spectra *to that laser*. moving the cursor over the spectra gives you actual numbers of % excitation & % emission efficiency.

You can use filters to test which filter is most suitable for your fluorophores.

This site is intended for FACS users, so you can choose from any one of a number of cytometers from BD. However, I think this site can be usefull for microscopists too.

 

EDIT: I found more sites like that, each with its own set of fluorephores –

Fluorescence SpectraViewer at Invitrogen (Life technologies) site.

Fluorescent Dye Spectra at U Arizona (only chemical fluorophores)

Evrogen Spectra Viewer at Evrogen (only a very limited list of fluorescent proteins)

 

There is also this Cell staining tool from invitrogen. The concept is cool, but I think it is a very limited tool and I don’t think it is very helpful.

Non-microscopy applications for fluorescent proteins

Here, I wanted to briefly discuss other applications of fluorescent molecules in biology, other than microscopy.

One simple use for fluorescent molecules is measuring the amount of emitted light by a fluorimeter. This can be used with different florescent dyes that respond to different biological aspects (e.g. calcium levels, cell viability, mitochondrial function, DNA replication, protease function etc…) and fluorescent or phosphorescent proteins (luciferase, GFP) to measure the amount of the expression of said protein fused to a protein or promoter of interest. This cannot be used at the single cell level but on cell culture, or cell lysate.

Fluorescence activated cell sorting (FACS) – this method uses flow cytometry, combined with fluorescence detection to sort cells based on different properties. These properties include cell size and morphology (based on light diffraction) and fluorescent properties, as designed by the researcher. In short, the cells flow through a very narrow tube and pass through several light and laser beams. The light scatter and the fluorescence of molecules excited by the lasers is detected by detectors found along the tube. Thus, one can count the number of cells that have this or that property. For instance, it is possible to count the number of cells that express GFP, and even divide them into groups based on fluorescence intensity (which is assumed to be equivalent to expression level of the GFP). For some applications, the cells are thrown away; for other applications, the machine can collect the cells with the required properties defined by the researcher (e.g. high fluorescence) so that the researcher can sort out undesired cell populations, and continue to work with only the desired cell population. Several years ago, I used such a set up to isolate cell clones that express high levels of a drug, which was correlated to high GFP expression.

Recently, a new technology was developed that combine FACS with microscopy, called imaging FACS. With this system you can take pictures of the cells that you are sorting. The resolution is high, but there are several useful applications.

Another use is as a biomarker to differentiate between different biological states. For instance, GFP could be fused to a promoter that is activated by high salinity in a certain plant. If that plant will sense high-salinity conditions, it will glow green.

A new commercial use is glowing fish. Other pets might follow…

Finally, GFP can be used as a publicity stunt to show biological capabilities (hence glowing fish, mice, rabbits, cats, pigs and monkeys. Soon – fluorescent humans).