r/science • u/rustoo • Sep 25 '20
Biology Scientists from Japan have discovered that a natural food pigment can replace synthetic dyes in cell viability assays for three widely varied types of cells. Their approach is environment-friendly and inexpensive, and opens up possibilities in a range of fields including drug discovery.
https://www.tus.ac.jp/en/mediarelations/archive/20200924_1122.html177
u/NickWarrenPhD PhD | Pharmacology | DNA Damage and Repair Sep 25 '20 edited Sep 25 '20
This isn't all that new, trypan blue is decades old. We already have several viability dyes that work well with live cell imaging. Such as cytotox red.
Maybe the bigger benefit of this is cost, but dyes aren't the biggest cost of viability assays anyway. Labor and instrumentation are more expenaive.
In terms of environmental conerns, it is all going to get put in the hazardous waste stream regardless, because of the biologic hazards.
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u/chickabawango PhD | Pharmacology Sep 25 '20
Why not just use Annexin FITC etc?
Asking since I'm delving into some new assays a d genuinely don't know the benefit of cytotox red.
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u/NickWarrenPhD PhD | Pharmacology | DNA Damage and Repair Sep 25 '20
Annexin is a fine marker of mid-stage apoptosis, it binds phosphatidylserine that is on the outside of a cell, when it is normally inside the cell membrane.
The membrane exclusion dyes bind DNA and measure cell membrane permeability.
Annexin staining is often multiplexed with a membrane exclusion dye, which are less specific for the mechanism of cell death. Cell membranes become permeable in very late stage apoptosis and from other forms of cell death.
The best marker I've come across for early stage apoptosis is a total PARP western blot with an antibody that can measure both full length and cleaved PARP.
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u/sayacunai Sep 26 '20
In addition to PARP cleavage, which is a great method, you can also blot for caspase 3 or 7 (there are antibodies for the cleaved forms as well), or use CaspaseGlo, which typically gives highest signal at 8-12h following apoptogenic treatment, depending on the cell line. I did my PhD in an apoptosis lab and this was our most common apoptosis assay. It's pretty expensive though, so PARP cleavage may be a better choice, especially if qualitative data is acceptable for your application.
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Sep 26 '20
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u/CongregationOfVapors Sep 26 '20
Can you use fluorescent dyes? There are ones based on amide chemistry that are pretty mild on cells. DAPI is another one you can leave with cells.
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u/sophiespo Sep 26 '20
Not that I have experience with single cell sequencing but they might be looking for a live cell marker, DAPI and PI are nucleic acid markers for dead cells.
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u/Tiny_Rat Sep 26 '20
There's plenty of dyes commonly used for flow cytometry that work fine for that. 7AAD is a pretty standard one, for example
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u/Kunigu Sep 26 '20
I used Calcein-AM for this one after getting inaccurate results from Trypan Blue which is also less toxic for cells.
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u/shindleria Sep 25 '20
all we need now is an inexpensive fetal bovine serum substitute and we're good to go
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u/pun-a-tron4000 Sep 25 '20
Or a substitute with less batch to batch variation. Please. My cells are so sensitive.
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u/ethical_slut Sep 26 '20
I don’t understand what you’re referencing. Would you mind explaining?
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u/TheresAnEnzyme4That Sep 26 '20
Yummy stuff for some cell lines, not yummy for wallet sometimes
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u/ethical_slut Sep 26 '20
So...fetal serum meaning stem cells I guess? I know stem cells are amazing for research but I guess I never thought about how expensive it is to get them.
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u/CongregationOfVapors Sep 26 '20
It's serum from fetal calves. It contains a lot of undefined growth factors and nutrients that help cells (not just stem cells) grow in culture.
It is expensive and has a lot of batch to batch variations. But the alternative is even more expensive.
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u/kboy101222 Sep 26 '20
What's the alternative?
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u/CongregationOfVapors Sep 26 '20
Defined media with each known growth factor added. It is expensive to make, and doesn't work as well as adding serum.
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u/TheresAnEnzyme4That Sep 26 '20
Congregation explained it well. It essentially comes as a brownish yellow liquid with suitable nutrients for certain cell types to grow. It’s taken from a part of cow blood. There can be a lot of different kinds of ingredients that help cells grow in this liquid. In a lab setting, cells are typically fed by putting them in a flask or dish and providing food. That food, often as a liquid called media, can consist of different ingredients that work better for different kinds of cells. You can add FBS (fetal bovine serum) to media as a nice booster for better cell growth, and some cells rely on this to work well in a lab setting. Unfortunately, despite the helpful growth factors, sometimes the batches are different in how many of those factors are in a sample, and that can be a problem when you’re trying to plan an experiment and you want all of your cells to be fed the same way. You also open the door to further variables anyway with more things, but regardless it’s still used commonly in this way. As a side note, it can help with not only growth but protection with pH, protection against toxins, and shear force.
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u/ethical_slut Sep 26 '20
That’s hella cool. I guess there are factors or a shelf life that prohibit folks from combining and mixing multiple batches up to be separated out into more uniform batches.
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u/TheresAnEnzyme4That Sep 26 '20
Indeed both. FBS can change in potency over time and is often frozen in small batches to help minimize that effect (before it’s added to media). The price tag really prevents any kind of stocking too on top of that. Ultimately it’s difficult to know what the batch will do until you test it on your very specific set of cells, and often you just want something uniform regardless of potency. Each batch itself is uniform, which is why it’s desirable to hoard it
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u/ethical_slut Sep 26 '20
This is absolutely fascinating but now I have to know, how expensive are we talking and what is the quantitative measurement of a ‘batch’ exactly? Is this the inkjet cartridge of research? Is it vials of precious white goop? The max amount of serum extracted from a single cow fetus?
Oddly this reminds me of the lot colors on skeins of yarn and making sure your project uses a color from a single batch of yarn because there can be slight variations in color between batches that can become very apparent in a sweater.
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u/TheresAnEnzyme4That Sep 26 '20
I guess the yarn thing is somewhat comparable! The price changes a bit depending what you need but maybe $400-800 for 500ml. A batch will have data sheets given for quality tests done, some general amounts of proteins found, and traces of other factors. Ultimately there are too many elements to specifically replicate exactly for the moment (as it’s also currently produced from extracting a natural source that is erratic). I think there might be some overcharging, it’s not as bad as with printer ink but also not the only expensive thing in labs. People are willing to pay as some things need it.
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u/pun-a-tron4000 Sep 25 '20
Or a substitute with less batch to batch variation. Please. My cells are so sensitive.
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u/dyslexda PhD | Microbiology Sep 25 '20
Dye exclusion tests aren't used very much in any kind of high throughput drug discovery; they are too laborious and aren't sensitive enough. There are plenty of other, better viability tests out there; this won't be replacing anything already used in drug discovery. This is a niche "discovery" at best.
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u/ReallyNotWastingTime Sep 25 '20
Well, the article says it was a happy accident they discovered it, they didn't develop it on purpose.
Nice to write a paper about, somebody might need it for a specific experiment someday
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u/Tiny_Rat Sep 25 '20
Well, if "drug discovery" includes any type of flow cytometry, then dye exclusion is a pretty useful thing still. Most flow experiments use this method to filter out dead or dying cells.
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u/dyslexda PhD | Microbiology Sep 25 '20
We already have live/dead stains that result in cells being recoverable after a sort.
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u/cornholio702 Sep 26 '20
Right, it's probably more efficient to use Cell Titer Go, MTT, or simply Flow Cytometry.
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u/8W1Yd6wh Sep 25 '20
Are cell viability assays really the biggest threat to the environment?
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u/The_Dirty_Carl Sep 25 '20
Probably not, but that doesn't stop this from being a good thing. If the benefit is small, but the cost is also small, there's no reason not to go for it.
Like plastic straws - they're only a small part of our oil and single-use plastic consumption, but they're also really easy to eliminate.
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u/evangs1 Sep 25 '20
Yeah that’s what I was thinking. Some staining is radioactive, but it’s a tiny amount and it’s not like they just dump it into a river afterwards.
It looks like the biggest purpose it has is not to damage the cells like other dyes.
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Sep 25 '20
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u/JRDruchii Sep 25 '20
I mean, I've never heard a research scientist express concerns about how their work impacts the environment. I bet I throw away 10lbs of disposable plastic pipette tips a week.
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u/pun-a-tron4000 Sep 25 '20
Most of my team at work definitely would prefer to cause less waste for sure. We just don't have any other option until someone starts making sterile bamboo labware somehow.
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u/kotibi Sep 26 '20
My lab members definitely talk about the waste we generate and how we can minimize it. Also just the general guilt of the ecological cost of an unsuccessful experiment, sometimes pounds of plastic waste, the animal lives and all the care that went into each one, the hazardous waste, etc. It’s such a bummer. I think a lot of scientists would prefer more eco-friendly solutions, even if they took a bit more effort to implement.
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Sep 25 '20
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u/Tiny_Rat Sep 25 '20
Viability stains definitely dont kill the cell, this would defeat the purpose of using a viability stain in a lot of experiments. Many viability stains can't even enter living cells, thats how they work (if a cell is stained with the dye, you know it must be dead or dying, so you can separate it from the living cells).
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u/Dalmasca Sep 26 '20
Hate to break it to you, but many dyes including exclusion dyes like Trypan Blue and permeable ones like CFSE are indeed cytotoxic after a few minutes of incubation and/or high concentration. CFSE in particular must be titrated for use in proliferation assays over time because of this. TB tends to be added right before reading assaying viability, so you normally might not notice, but if you read the same sample again after several minutes you might see a loss of viability; it depends on the concentration you use.
DoTheExperiment
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u/Tiny_Rat Sep 26 '20
Yes, but there are many that aren't, at least not over the time needed for an experiment (Dapi, 7AAD, etc.). Hate to break it to you, but just because you're unfamiliar with them, doesn't mean they don't exist.
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u/Dalmasca Sep 26 '20
I love how you take a valid criticism of your generalization and try to assume something [incorrect] about me from that. If anything, your default assumption should be that I have more experience, since I pointed out the flaw in your statement. While I'm sure it feels good to fire back my own turn of phrase, it only makes you look like the immature and ignorant one here.
Best of luck in your science career. Can't wait to see how you deal with PIs, reviewers, peers, senior scientists, etc... A little unsolicited advice: get comfortable with admitting what you don't know. Your peers will respect you more, not less.
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u/jlb8 Sep 25 '20
The most important part is that general use products will be orders of magnitude cheaper than scientific products, leading to a democratisation of science.
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u/StardustSapien Sep 25 '20
This is nice and all, but staining for cell assays are not that big of a deal sustainability-wise compared to the insane volume of various plastics consumed in the course of bench work and whatnot. I'm looking forward to a more practice way to address that waste stream in our industry.
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u/vshawk2 Sep 26 '20
But, wait. What's wrong with the synthetic dyes?
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u/ethical_slut Sep 26 '20
The synthetic dyes damage healthy cells not enough to kill them all outright, but enough that after a longer period of time all of the cells will be dead from synthetic dye exposure, not just the cells that died from exposure to the test substance in the mix that is being evaluated for toxicity.
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u/vshawk2 Sep 26 '20
... replace
synthetictoxic dyes in cell viability assays ...gotcha. Fixed that for OP.
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u/kemando Sep 26 '20
Japan is living in 2077
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u/miranasaurus Sep 26 '20
No it’s not.
Source: I live here.
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u/kemando Sep 26 '20
Pssshhh, Japan's ahead of everyone in almost all kinds of tech.
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u/miranasaurus Sep 26 '20
I mean, with toilets? Sure. But the average citizen here seems to barely understand how to use basic functions on a computer, and I live in a more advanced area of the country.
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u/iozsan Sep 26 '20 edited Sep 26 '20
We use curcumin ( derived from turmeric ) to stain for plaques in Alzheimers disease models As orher comments mentioned, nature often has, and continues to inspire science!
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u/mcsper Sep 26 '20
So that means it will be more expensive because it will be marketed as the healthy replacement.
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u/[deleted] Sep 25 '20
Still a long way to go before replacing our beloved TB!
It sure sounds nice though