Today we discuss a cell viability assay called MTT assay. We have graduate student Marzia Pendino joining us to walk us through this technique. Thank you for joining us, and I hope you keep listening.
Today we discuss a cell viability assay called MTT assay. We have graduate student Marzia Pendino joining us to walk us through this technique. Thank you for joining us, and I hope you keep listening.
Hey everyone to another episode of nerd RX podcast and I'm your host Barkha. Today we have Marzia Pendino and she is going to talk about MTT assay. Welcome, Marcia to the show.
Marzia Pendino:
Hi Barkha thank you for inviting me
Dr. Barkha Yadav-Samudrala:
I'm so happy that you could be here. So before we go into the topic, why don't you introduce yourself to our listeners?
Marzia Pendino:
Yeah. So hi, I'm Marzia Pendino, as you said, and I'm a chemist pharmacist. And actually, I'm a third year PhD student in University College Dublin, in Ireland. And my project is funded by iurc and rectal cancer research charity, based in Ireland. And my research is focused on the evaluation of the durability potential of cannabinoids in uveal melanoma. So actually, we all know Noma is a rare cancer. But it's one of the most intraocular malignancy in the adult eye. And it arrives in the eye, but then he spread on the liver. So right now, there is no standard of care for this patient. So it will be good to figure it out. If cannabinoid could be like, potential treatment for actually this patient, the bad thing is that a few patients has no symptoms at all. But other one present for example, our change of their race, color flesh of the light, so it's important that they take care about this and they check with the ophthalmologist as soon as they can.
Dr. Barkha Yadav-Samudrala:
Well, thank you so much for that introduction. And you have an amazing background with chemist and pharmacist and now research I am also a pharmacist and a chemist in our research. We have something Yeah, we do. So, let's talk about MTT assay and why did you pick this topic and what got you interested in it?
Marzia Pendino:
Yeah, so, as a as a researcher, so, and as a cancer researcher, so, what I did is try to figure it out, which will be the best hasay for starting and to have like a drug screening or regular screening for my project. So and I pick it up these MTT assay that is used to measure like the cellular metabolic activity as an indicator of the cell viability. So, is simple Komal colorimetric assay that is made in the reduction of yellow transcellular sold to a purple for Madonna crystals by of course, the metabolically active cells. So, what happen is that the viable cells contain oxido reductase enzyme which reduced the MTT to the format's and increased cell okay. So, it's actually really simple to conduct and what you can see is that you have a change of color in the plate. So, briefly is really simple. So, any researcher even the newer, the new researcher can perform this assay. So, you need to use a 96 well plate and you can see the cells in the world and then you need to treat the cells with drug treatment that you wish to to use and after the time treatment that you decide to pick up, there could be four hour 24 hour 96 hour based on different projects actually. You can then perform the MTT assay that consists in actually remove this drug treatment and then add these MTT solution for like maximum four hours some of the researcher use these for like two hour an analysis and then after that you can see the format's on the crystals. They are like purple. Of course you need to solubilize these crystals and we usually use DMSO and then you get Read the plate in the spectra for the meters at specific like nanometer that could be between 500 at 600 Actually, but by your high you can actually see different color of the of the well of course, the viable cells will have really strong purple color the non viable cells will be more fainted really close to the yellow like
Dr. Barkha Yadav-Samudrala:
yellow okay. So, is it like concentration dependent?
Marzia Pendino:
No is the E depends of the viability of the cells, okay, because the variable cells will have these oxidate these oxy reg doctors, enzymes, active cells that will show like the results.
Dr. Barkha Yadav-Samudrala:
Okay, so, for example, I have a drug and I know, this works against cancer cells that I can use in different concentrations right to check with the MTT assay to see the cell viability and get a dose response curve.
Marzia Pendino:
Yes, it's actually what we what you have to do. So, you can treat the cells with different concentration, and then you will check which is the concentration of the drug that statistically reduced the cell viability of the cells. Of course, you don't want completed the cells that otherwise you can not see any results, but you Your aim is like, find a concentration that will impact the viability but will not kill the cells totally
Dr. Barkha Yadav-Samudrala:
correct. Okay. Okay, got it. So how long does it take the entire experiment from start to finish?
Marzia Pendino:
It really depends of the time point. Where do you want to check the viability? So for me, I use for example, 96 hour of treatment. So everything is based on your time treatment, but then MTTR Sabir say it lasts from three to four hour, four hours, but you don't need to actually work for all the time, because you need like,
Dr. Barkha Yadav-Samudrala:
time incubation. Yeah. Correct. Yeah. So so how do you figure out that treatment time, like you mentioned, for you, it's 96, but it would be different for someone else? So how do you figure that out?
Marzia Pendino:
Yeah. So something that you can check before is like in literature, because a few other researcher for example, have done the same assay in the same cell line, for example, and you can like note, like this, or, for example, they use the same treatment in a different cell line, it really depends of the disease that you are studying, if you know that there is a specific time point that you should follow. And I will always recommend to check in the literature. Okay. Yeah, so this is actually an assay, an in vitro assay that you can use as a starting point for doing drug screening. And to have like, a few data about like the viability of the cells in particular for me, they are like cancer cells. But something that I'm doing right now is like something related with the tumor patient samples. So I was starting with the, to the like, cells, and now I'm moving to 3d, like, type of experiment. So yeah, so actually, what I'm doing is I'm receiving biopsy. So when I talk about tumor patients sample are actually biopsy. So they are part removed as part of scheduled surgical removal from patient that has these tumor, and it will actually help me to test preclinically the drug that in vitro was already tested. So I started from an in vitro assay. And now I moved to a preclinical assay. I and that will give me results more related to patient instead of only cells.
Dr. Barkha Yadav-Samudrala:
Okay. So you have already taken further this assay in your race. Yeah,
Marzia Pendino:
yeah, so actually, what I'm doing is I'm receiving the samples from the hospital. So patient got where normal check their hospital. And of course, the bad point of these is that sometimes the biopsy so the tumor is so small that they need this tumor like for normal check in the hospital. So sometimes we're going to really use these biopsy for our treatment. But of course they need to, for further like analysis and for further like detection in the hospital. But most of the time, we can get the samples and we can what we do is we divided in different pieces, and we place in the six well plates with the treatment that we want to does, of course, as for the MTTR, say, I will use that control. And then I will treat these pieces of two more. And that's important because actually the micro environment in this case is one of the most important things. Because since is that you want from a patient, there will be as soon as I treat the samples, there will be like a lot of factors that will be released, and can help me to figure it out more about the disease about like what happened and which are this sacred on factor that are released from the tumor. Something that yeah, something a bit like Twiggy is the fact that of course, I need we need a tickle application for performing this experiment. Instead with MTT you can actually perform the assay with no application with no medical application since they are like cells. In this case, the process for be approved as in order to like collect these, these tumor patient samples is a bit long, especially here in Ireland. But we actually got from a few Ospital like this ethical obligation. And yeah, he does pass to actually reach and reach patient and clinician. And because we need both of them to add like researcher to bring like new treatment and to study the disease. I mean, that's something we aim all the time. Yeah.
Dr. Barkha Yadav-Samudrala:
So it's the there are there any like when we talk about MTT and cell viability assays, are there any alternative techniques to MTT to get the same results.
Marzia Pendino:
Um, there are other like, assay that you can use. But there is something that you can use also in cancer cells, that instead of checking the viability of their cells, you can check, actually that proliferation, so, how the cells proliferate after they were treated with my case with an anti cancer like treatment, and it's really similar to them MTT assay, but she's way easier, because esign you need to see the cells in our six well plate and you need to treat the cells at different time point. And then you need to remove the treatment, but you need to leave the cells in the six well played with completed media, so with FBS or pen strep or whatever, for at least like a number of days. So in my case, I use for example, a days and after these eight days, you can see that the cells are not single cells, but the cells revive after the treatment they are created like clone. So they are going together at Clay This small like.on The world. And you can check by like scanning the six well plates, which is the amount of clone that are formed after the treatment.
Dr. Barkha Yadav-Samudrala:
Okay, interesting, like you're going from viability to proliferation that is, that is really cool.
Marzia Pendino:
Yeah, he's Yeah, he's really.
Dr. Barkha Yadav-Samudrala:
And I think you mentioned this previously that these techniques are very user friendly, anyone can quickly learn these things, right?
Marzia Pendino:
Yeah, that's, that's something really important, especially if you are a new researcher figure it out, which is, which are the most easiest like technique that you can use, especially at the beginning. And I think that these two are say, are really friendly use and it doesn't the cost is really like, is not expensive at all. So I mean, you need to buy Of course, the, the 96 or the six well played. But I mean, you need to buy the empty team, in the case of them DTSC but at the end, you don't need like in like to spend a lot of money. So it just really easy to perform. Yeah.
Dr. Barkha Yadav-Samudrala:
So what are the advantages and disadvantages for entity?
Marzia Pendino:
Yeah, actually for MTP so is our niZi hasay has I mentioned before, and is actually cheap or cheap, per se, and you can really easy results really good results with and then replicate three. So that's that's really good. If I have to mention the disadvantages, it will be for example, that you are the beginning you need to have figured out and test different concentration of your treatment before reaching the right reduction in viability and the troubleshooting the beginning of the protocol because you need to figure it out, which is the time of incubation with MTT solution and the reading of the plate. I mean, it can happen that sometimes based on the treatment that you are testing, you can kill the cells actually you have no results available. But I mean it's trouble soothing is science. We get used with
Dr. Barkha Yadav-Samudrala:
Yeah, I don't think you can do any experiment with at least a few failures.
Marzia Pendino:
No, not at all. I don't think but we need this to grow. Yeah.
Dr. Barkha Yadav-Samudrala:
Yeah. Okay. So, my last two questions that I asked everyone is do you have any fun fact for MTD acid
Marzia Pendino:
Yeah, I mean actually, there is something so since since I say show different grades of purple the viable cells will be will have like strong purple the less viable will be fainted powerful of yellow in the case of of non viable cells. For this reason, researcher called this assay like 50 shades of purple.
Dr. Barkha Yadav-Samudrala:
Oh my God,
Marzia Pendino:
because really, you can see by your hide the results before reading and spectra
Dr. Barkha Yadav-Samudrala:
Oh, wow. That's yeah. Yeah. Well, and my last question to you is, would you can you suggest any article or protocol related to MTT assay so that I can link that down in the description for our listeners?
Marzia Pendino:
Yeah, so, actually, these MTT assay made for the first time by theme Mosman. And I think he published our paper on the journal monumental neurological metals in the 83. And yeah, the title will be rapid colorimetric assay for cellular growth and survival, application to proliferation and cytotoxicity assay. So, and he mentioned all the, in the material in the matter, the cells that he use, and the colorimetric assay that he performed. With MTT, so it's well explained. And I suggest if you want to actually have a look at these also for how to validate the results, and I mean, it's really, really interesting.
Dr. Barkha Yadav-Samudrala:
Well, okay, thank you so much. And with this, we are going to end today's episode. Thank you so much, Marcia, for being with us today.
Marzia Pendino:
Thank you Barkha. And see you soon.
Dr. Barkha Yadav-Samudrala:
Yeah, see you soon. And listeners, please tune in next week for another episode of Nerdrx podcast and in meanwhile, if you have any suggestions for topics, or if you would like to come on this podcast and shared with us something you have, please email me at Barkha@Nerdrxpodcast.com. And remember, it's good to be a nerd, bye.