What I do at Work

‘What do you do at work?’

A rising tingle of stress creeps up whenever I try to answer this question.

Heck, attempting to write this piece is nerve-racking.

Major Disclaimer: Any opinion that I happen to express here is solely my own and not of my employer’s and colleagues.

Well, for starters, I work in a core facility lab, AKA a service lab. I am part of a team that helps researchers get a certain niche thing done in order for them to conduct further experiments.

That niche thing is called ‘Flow Cytometry’.

Did your mind just gloss over that? No worries!

Well, here’s the analogy that I usually give family and friends who ask:

Imagine a bowl of multi-coloured M&Ms, with different flavours: milk chocolate, dark chocolate, peanut butter, whole peanuts, etc.

The machine I operate helps to separate these M&Ms by colour and flavour! Of course, in reality, these M&Ms are cells. These cells can be cultured (grown from a dish), from tissues or organs, from human and/or animals, depending on the experiments conducted by the researchers we work with.

My explanation usually ends there.

But here’s a tad bit more into what this technology does and *surprise surprise* how it plays a huge role in immunological research. *ignores the gloom of the pandemic*

Da heck is ‘Flow Cytometry’?

I will try my very best to simplify things here.

‘Flow’ meaning, well, flow.

‘Cyto-’ here means ‘cell’ and ‘-metry’ is ‘measure’.

So essentially, flow cytometry means putting single cells in a straight line, suspended in liquid and make them flow through a flow cytometer; the instrument. Think of the pearls (boba) that go up your straw when you’re drinking your favourite bubble tea. They go up your straw one by one, driven by pressure changes created by sucking (*tries to maintain a straight face*). Same thing.

#professionalism

With the cells going through the machine in a uniformed flow (*cue drinking game*), there are lasers at a certain point that act as an interrogation point. Like a customs check.

A laser hits a cell and the cell then refracts or scatter the light that passes through, over or under it. Like when shining a torchlight at a glass of water in the dark and the glass of water becomes a lamp.

Light scatter

Any light that makes it through, over or under the cell on the other end, gets collected by a photodetector, which converts the light into electrical signals.

These electrical signals then tell us how big the cell is and how complex the insides of the cell is. And if the cells are sticky and stick together (like some pearls do), the light collected can also tell us whether or not it is a single cell or doublets.

So that’s the very, very simplified, watered down explanation of this technology.

I first learnt about it during my Diploma days and it was pretty fascinating. But how? How? How do you separate them by colours or flavours? Like how does the machine know about the different M&Ms that flow through?

Taking it up a notch: Colours

First things first, the cells themselves aren’t actually coloured. If they were, we’d be walking unicorns. If you want to separate cells based on more than just size and structural complexity, then you’d have to stain them. Again, we don’t actually stain the cells with dye…okay we kinda do.

Hear me out.

For one, we can put dyes that only stain dead cells. Since dead cells tend to be porous (have holes poked into them), they’ll take in the dye which gets ‘activated’ by a laser. Live cells are pretty fussy with what they allow into them and thus, will not get stained.

Live dead stain
Pink cells are alive and vibin’, while the blue-gray ones are as dead as nails. The blue mist here is the viability dye which will get washed away after about 20 mins of incubation/staining. The wash happens so that we have a lower background signal later on. It’s like choosing a plain background for your desktop wallpaper so you can actually see your apps.

Literally all that glitters glows is not gold.   

Oof.

Anyhow, from there, we can start to stain cells for type: are they human? Which organ are they from? Which tissue of that organ? Subtype? Etcetera.

That brings us to a term you hear get thrown around a lot lately: antibodies.

These are proteins that simply bind onto something they ‘hate’, thus the ‘anti’ in antibodies. There are antibodies out there in the market that bind to specific proteins and these antibodies are also tagged to a fluorophore. A fluorophore is a particle that will emit light when ‘activated’ or more accurately, excited by a laser of a certain wavelength. I shall conveniently leave out the physics.

This antibody and fluorophore pair then become known as our ‘stain’.

When you add this ‘stain’ into a tube of cells, the antibody portion will bind to its hated specific protein present on the surface of the cell! Thus, when you pass the cells through the machine, only cells with that surface protein + antibody + fluorophore will light up in say, red.

Antibody complex
Its like my graphics regressed. Like the stock market.

So if you change the antibody to target another protein and tag it to another colour i.e. green, you can find out which cell has two colour signals (red & green) and which cell has only one (red or green) or none. And you can also find out roughly how much of each protein is present on the cell.

These surface proteins are also known as markers; which in this case are proteins unique to a particular cell type or subset(s). Other examples of biomarkers can also be in the form of genes, molecules, etc.

Flow cytometry & the immune system

Your immune system, for the uninitiated; is made up of your white blood cells…and your skin and other tissues and organs but for the purposes of flow cytometry, let’s focus on the blood.

Needless to say, these are the cells that actively patrol your body for things that shouldn’t be there. Things like bacteria, viruses and even, cancer cells!

And because these cells have tons of things to do, they specialise! The work is split up between themselves and thus, you get different types of white blood cells. If you cut yourself, the neutrophils will be there. If you have a parasitic infection (yikes), eosinophils will come by and dissolve the naaaaasties.

Eww. But ooooooo.

You get the idea. If this interests you, may I direct your attention to this anime that my colleagues and I love, coz we nerdy AF.

It’s an anime! About your immune system! And the main characters are a red blood cell that delivers oxygen in a freaking carton box as if its FedEx. And a neutrophil?!!? I LOVE THIS. There’s a reeeeeally strange sexual tension between those two characters BUT HEY. Ship ‘em if it makes you happy. *shrugs* And the platelets are! so! cute!

Okay.

Thus, in order to determine the health of an immune system or if you’re a researcher studying only one cell type out of the many; flow cytometry is a good way to have a zoomed-out, overall view of a subject’s blood and/or immune system. Things like percentages of each cell population can be determined, kinda like a full blood count. But inclusive of sub-sub-sub populations. We have maaaaaaaany types of immune cells in our blood and our tissues.

Immune cells
Non-exhaustive! P.S. Someone tell me how to up the image quality from Powerpoint.

Yeeeeps.

Each cell type has a unique combination of markers and therefore, we can stain a blood sample with a cocktail of antibody dyes. This cocktail is known as a panel and could look something like this:

Marker Dye/Fluorophore Present on…
Live/Dead AmCyan
CD45 BV605 All immune cells
CD3 Pacific Blue All T-Lymphocytes (T-cells)
CD19 PE B-Lymphocytes (B cells)
CD14 FITC Macrophages
CD66b APC Granulocytes

(e.g. Neutrophils, eosinophils)

Disclaimer: If you’re a seasoned flow cytometrist, I came up with this on the spot, don’t hate me for my dye choices *hides head in sheath tank*

The complexity of a panel heavily depends on the research question and the specifications of the flow cytometer.

Getting an overview of each sample allows researchers to do something like comparing stimulated versus non-stimulated states. For example, vaccine responses. You’d expect certain cell types to increase or decrease in number after administering a trial vaccine. If there are no changes as compared to a control/baseline reading, then it is most likely that the vaccine did not work or perhaps another method has to be used to detect changes, e.g. gene expression, proteins.

And the best part about using flow cytometry, you can actually sort the cells you’re reading. A flow sorter allows analysed live cells to be collected and used in further experiments such as DNA or RNA extraction, cultures, western blots, etc. Meaning, you can walk away with a bowl of just blue-coloured milk chocolate M&Ms. And then use them to decorate a cake! Or maybe you just love blue milk chocolate M&Ms.

I know I do.

Wew, that was an essay

So this was a suuuuuper duper in-depth answer to ‘what do I do at work?’

It is almost the length of a short-report and possibly the longest blog post I’ve ever written.

Me mentioning vaccines earlier on would have probably triggered thoughts about the pandemic. If you’re wondering more about the process of vaccine research and development, here’s an interview I really enjoyed. It’s 40 minutes long, but Dr Kim explains it so simply, its beautiful. Like it made me wish that he was one of my immunology lecturers back in uni.

 

Enjoy and till the next one, take care!

Posted by

Biological Science graduate. Full-time working adult. Loves planning stuff. And lists. Appreciates all things artsy-fartsy. Sings in the choir, but can't do solos.

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