Automated slide-free cell counting with CellDrop

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Hi, everybody. Thanks for joining us today, and good morning,

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good afternoon, and good evening.

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I can see from the attendee list we've got quite a range of

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locations for where people are from.

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So whatever time of day it is, I really appreciate you joining us

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for the webinar today.

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So the title of today's webinar is Count Cells Without Slides:

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Automating and Optimizing Cell Counting.

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My name is Andrew Jones, and I am the market development manager

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with DeNovix.

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I'll be moderating today, and presenting will be

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Cory Pizak, who is one of our application scientists with

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DeNovix, and he'll be taking us through how to standardize cancer tissue

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culture, primary cells, nucleic acids, and other complex

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samples on the CellDrop automated cell

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counter.

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The webinar will look something like this.

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We will have an introduction to the technology of

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CellDrop, which will take about 10 minutes, just for those of you who aren't

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familiar with it already. We'll then switch to a live

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demo. So Cory is based in our Wilmington, Delaware headquarters,

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and he's in the lab at the moment, and he'll have some live cells that he's going

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to run to show you some examples of how to do that on the

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CellDrop. That'll take about 10 minutes.

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And then we'll also

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finish with a Q&A session.

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So for the questions and answers, you will see in the Zoom

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interface, you have a Q&A option in there.

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During the course of the webinar, if you have any questions, please feel free to

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add them to that

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section. We'll monitor them, we'll answer them as we go along,

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and any of them that are relevant to the wider group, we'll

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put those to Cory when we do the Q&A.

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So before we get started, I'll just do a quick introduction to DeNovix in case

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you're not familiar with our company.

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We were founded in 2012 by Fred Kielhorn, and Fred was one of the

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original founders of NanoDrop Technologies, if you're familiar with

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that technology.

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We're based in Wilmington, in Delaware, in the US,

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so all of our product development, all of our application support, all of

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our manufacturing service, is all based at our HQ

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in Wilmington.

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We're the inventors of multiple award-winning instruments,

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and we're proud to have some of the most highly reviewed

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instrumentation on the SelectScience web portal, which is where we choose to

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collect our reviews.

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We have over 12,000 citations and 1,000 plus reviews from

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researchers around the world, which we're very proud of.

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We like to think this is a combination of great instrumentation and

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our focus on providing the best technical support we

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can. So we're laser-focused on being one of the best in the industry for

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supporting you.

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Our product portfolio has three main pillars.

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So our original product line was the DS series,

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and that's a very successful micro-volume spectrophotometers,

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fluorometers, and combined instruments.

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The CellDrop automated cell counters are going to be the focus of today's show.

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So I won't talk too much about those because Cory will give you the introduction.

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And we're also very excited and very proud to announce our latest

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product that we have just launched, which is called the Squid Pipette.

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And the Squid Pipette

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is a unique instrument. It's the first electronic pipette that

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allows you to aspirate the full volume of one to

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1,000 microliters in a single device.

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So if you're interested in that, lots of information on our website, but that's not

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the focus of today. But feel free to reach out for more

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information on that if you're in the market for a new pipette.

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Today's speaker is Cory Pizak. Cory is a

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member of our application support team, based in Wilmington, in

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Delaware. As you can see, Cory is in our lab at the moment.

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Cory has a master's degree from Villanova in biological

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sciences and a specialty in molecular genetics

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and immunology. And Cory's focus day-to-day is

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largely just providing technical and application

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support to our install base and to

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anyone currently running free trials on our

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instrumentation.

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So with that, I'm going to hand over to Cory.

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Thank you, Andy, for that introduction.

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Before we begin, I just want to tell a very quick and brief story.

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So during my graduate studies at Villanova University, my

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research focused around macrophages, and I began

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by counting them manually with a hemocytometer, and I really wanted something

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to streamline that process because, let's be honest, it is a very

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laborious process. Luckily, my neighboring lab has

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a CellDrop, and I was able to use it.

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And I don't want to say this instrument's the reason why I finished my project

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three months early, but it had a big part in it.

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So enough about me. Let's get into why you're here.

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So you may be asking yourself, how does the CellDrop count without slides?

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First and foremost, it's the direct pipette technology because

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it eliminates the need for expensive classic slides.

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This instrument has dual fluorescence and brightfield technology,

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which is great for trypan blue and acridine orange and propidium

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iodide counting.

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It has a suite of apps for multiple workflows and an

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HD touchscreen, just like your smartphone or tablet.

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And finally, it has flexible connectivity to export your data in

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multiple ways.

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Now to dive deeper into the direct pipette technology, the

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upper and lower sample surfaces are optical grade sapphire

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surfaces, so they're very robust and scratch-resistant.

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And you could think of the CellDrop as a hemocytometer microscope

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hybrid, and you'll see more during the live demo.

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So as the animation shows, once you pipette your

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sample between the two surfaces, surface tension holds the sample

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between the chamber. Now, we do have three chamber heights that are controlled by a

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precision screw, which I'll talk about next.

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So altogether, this technology is easy to keep clean with no

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sample carryover and no calibration required.

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So there are benefits of the direct pipette technology.

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First and foremost, it's environmentally friendly because again, you do not

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need to dispose of any plastics. Now, a few

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benefits of the chamber heights. So one, there's no need to

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dilute or concentrate your samples because we have a

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wide density range of 700 cells per milliliter all the way up to

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25 million cells per milliliter. And we also have a large cell

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size range from four microns to 400 microns.

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Now, I would like to focus on the chart at the bottom of the

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screen. As you can see in that middle column, 100 microns,

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that's our standard chamber height.

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But to the left and to the right of it, we have two other chamber heights

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for higher density samples and lower density samples.

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Now, the CellDrop has two modes. We have bright field, which is great

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for clean tissue culture samples, which you can use trypan

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blue or Erythromycin B for viability assays.

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We also have dual fluorescence for primary cell samples, or

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if your cells have a funky morphology.

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This is great to use fluorescent assays like acridine orange propidium iodide,

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or if you have a GFP tag inserted in them.

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Now, I want to talk a little bit about the assays that I'm going to

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demonstrate on the CellDrop. First is the bright field viability

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assays of trypan blue and Erythromycin B.

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Now, they both select,

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they stain dead cells in a dark color, and they permeate into

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dead cells and bind to negatively charged molecules.

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And they're very commonly used in manual cell counting.

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And if you haven't heard of Erythromycin B, don't worry, it is new,

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it is non-toxic, and it is better than trypan blue.

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Now, let's switch gears to fluorescence counting with acridine

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orange propidium iodide, which is commonly referred to as

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AO/PI, and I will call it from here on out.

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So they are nucleic acid binding dyes. Very, very specific.

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So AO permeates the cell membrane of both live and dead

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cells, and it fluoresces green, so you can think of green

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live. And then PI cannot permeate live membranes and only

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enters compromised dead or dying cells, and fluoresces

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red. So red dead. And this is because there is a fret

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quenching in dead cells that causes PI to absorb the AO

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fluorescence signal, ensuring precise counting.

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So you may be asking yourself, when and why would I use fluorescence

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counting? So again, for primary samples like the top image

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that are littered with red blood cells, this

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stain is great because trypan blue is a blanket assay and

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will non-specifically bind to debris and non-nucleic cells

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such as red blood cells. Whereas AO/PI will specifically

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bind to live and dead nucleic cells and not

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staining red blood cells.

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So I want to talk about a little bit of the data between the two assays

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and when to use them. So the top graph are Cho tissue

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culture cells, and as you can see, there is a one-to-one

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comparison with trypan blue and AO/PI.

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But the bottom graph are primary PBMC samples, and as you can

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see, trypan blue over counted by a factor of two when you're

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comparing them to AO/PI, and this just means AO/PI is a lot more

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specific.

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Hey, Cory. Okay, great. Thanks. So we've got a couple of questions that have come

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in already. If you do have more questions, as I mentioned, put them

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into the Q&A box, and we'll pick them up as we go along.

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Just a couple of questions. First one that we had in was whether the

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CellDrop has to be connected to the network in order to analyze cells.

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Yeah, great question. It does not, for a few reasons.

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The first one is all the data is stored on the instrument

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itself, and then you can always export via

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USB. So there's no cloud-based storage.

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And all the analysis takes place on the instrument as well, of course.

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The other question was why does Erythromycin B

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work better than trypan blue?

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Yeah. So Erythromycin B is superior to trypan blue, like I said, because

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it is non-toxic, it has a longer lasting stability when

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stored, and enhanced optical contrast for automated counting

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systems just like the CellDrop.

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Great. Thanks, Cory. That's all the questions we've got for now.

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Wonderful.

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Thanks.

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So now I'm going to be switching to a live demo.

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I'm going to be using the CellDrop FLI or the FLI, as we like to

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call it. We'll be doing three counts today.

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First one is going to be Jurkat with trypan blue.

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Then I'm going to switch to fluorescence counting.

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The second count is going to be Jurkat with AO/PI, and then primary

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PBMC samples with AO/PI.

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So here's an overview of the instrument.

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As you can see, this screen here is a seven-inch HD

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touchscreen. Again, just like your smartphone or your tablet.

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And we have the three apps right here.

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We have Trypan Blue, AO/PI, and Primary Cell

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AO/PI apps that we are going to be using today.

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So as we move up, you're going to see where you load your sample.

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And as you can see here, once you lift the arm up, we have both sample surfaces.

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So we have the lower one here, and we have the upper one up here.

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So to load your sample, you're just going to put the arm down.

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So I'm going to open up the suite of Trypan Blue apps here, and I'm going to

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select the Tissue Culture app.

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So now I'm just going to do

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a little bit of a mixing of our cells,

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mix them with Trypan Blue,

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and then I will be loading onto the instrument.

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And now I'm doing a one-to-one mixture of 10 microliters

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of cells, 10 microliters of Trypan Blue,

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and then I'll be loading on the instrument.

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And then to load, what you'll do is there's two notches on either

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side. You use them as a guide for your pipette tip up

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to where the sample surfaces meet, and pipette normally.

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So that's my favorite part. This is a live feed.

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Watching all of your cells go across the screen is my favorite part.

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So what we're going to do now is we're going to check the exposure.

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As you can see, we have different exposure settings, and there's going to be these

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information boxes on the bottom left of all of these windows just to

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show you if you're correctly exposed or not, and that's the same thing with

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the focus.

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Now everything is set correctly. I'm just going to press count.

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And within a few seconds, we're going to see not only a

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picture of the cells on screen, but we're also going to see some

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data on screen as well.

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So as you can see on the left-hand side, we have live cells per mil, dead cells per

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mil, total cells, and a viability percentage.

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And then if you move down here, you can see some nominal cells as well.

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So cleaning the instrument is very quick, very easy.

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You're going to take a Kimwipe, fold it up a few times.

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And once you lift the arm, data is saved automatically.

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And again, these surfaces are optical-grade sapphire.

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They're very robust. So you could put a little bit of pressure on them.

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You will not break them.

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And just like that, we clean the instrument, and we're ready for our next count.

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So now I'm going to be going to our AO/PI counting

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with our AO/PI app since we're going to be using Jurkats.

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And just like with Trypan Blue, I'm going to be mixing some

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cells with our AO/PI.

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Now, even though it's a fluorescent stain, a lot of people do have that

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question of, do I have to incubate my cells before I load them on the cell

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00:15:06.584 --> 00:15:10.504
drop? And that is no. We do recommend to mix them

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00:15:11.104 --> 00:15:12.044
and load them right away.

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And just like that, you're going to see your cells come across the screen again.

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00:15:22.304 --> 00:15:26.124
Now, with AO/PI, we have three different channels, just because it

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is fluorescent. We're first going to go on the green channel.

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We're going to check the exposure.

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Again, there's an information button here,

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and the focus in the green channel.

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Then we're going to move over to the red channel and make sure that the

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exposure is set properly as well, and only the

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exposure. Everything is set, and now I'm going to press count.

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And you may have noticed I did not change the focus and exposure settings,

254
00:15:52.464 --> 00:15:56.144
and that's because the instrument remembers all the settings that you

255
00:15:56.364 --> 00:16:00.324
previously inputted. So that means a very quick workflow

256
00:16:00.364 --> 00:16:02.624
that you don't have to change settings between counts.

257
00:16:03.384 --> 00:16:07.044
And just like the Trypan Blue app, you're going to have your live cells, your dead

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cells, and a viability percentage with nominal data.

259
00:16:11.164 --> 00:16:13.844
Now, if you need to gate on the fly, you can do so.

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If you touch this button down here on the bottom left, a graph is going to pull up,

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00:16:18.244 --> 00:16:22.024
and you can use this sliding bar to gate your minimum or

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maximum size. And then if you do so, you can apply those

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00:16:25.904 --> 00:16:26.884
gatings to result,

264
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and the live and dead cells will change.

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Again, cleaning just like the Trypan Blue app.

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We're just going to

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firmly press and wipe the top and bottom sample surfaces.

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So now I'm really going to show you why AO/PI is so valuable

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00:16:50.124 --> 00:16:51.584
because I'm going to

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do a primary cell count with PBMCs

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and whole blood.

272
00:17:01.824 --> 00:17:04.744
So again, just doing my one-to-one dilution.

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We're going to mix really quickly,

274
00:17:08.764 --> 00:17:09.464
and we're going to load.

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And as you can see on screen, there's a lot of darker

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00:17:21.264 --> 00:17:23.714
cells. Those are the red blood cells.

277
00:17:24.204 --> 00:17:27.844
And as you can see in the green channel, we do have a few green dots.

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00:17:27.944 --> 00:17:31.774
Those are our live cells. And in the red channel, you will

279
00:17:31.864 --> 00:17:35.844
see some dead cells. And then we're just going to go ahead and press count.

280
00:17:36.844 --> 00:17:40.694
And again, within a few seconds, we're going to get an accurate count of live and

281
00:17:40.764 --> 00:17:42.954
dead cell samples within the sample.

282
00:17:44.284 --> 00:17:48.154
So as you can see here, all of the darker cells are

283
00:17:48.184 --> 00:17:50.154
red blood cells, and they're not being picked up,

284
00:17:51.144 --> 00:17:52.304
but these green ones are.

285
00:17:53.174 --> 00:17:56.724
And if you just want to check and make sure which cells are live and dead,

286
00:17:57.404 --> 00:18:01.244
we have this channel button right here, which you can look at your cells in any

287
00:18:01.304 --> 00:18:04.064
channel that there is. So I want to look at the green channel.

288
00:18:04.664 --> 00:18:08.384
I could do so here and make sure my green signature is being picked

289
00:18:08.444 --> 00:18:09.064
up.

290
00:18:11.424 --> 00:18:15.284
And now if you want to optimize any settings, you could do so on the fly as

291
00:18:15.324 --> 00:18:16.904
well with this optimized settings button.

292
00:18:17.484 --> 00:18:21.374
If you change any of these parameters, you can count with the current settings

293
00:18:21.964 --> 00:18:25.444
And then a green box over here will pop up saying Save settings to

294
00:18:25.464 --> 00:18:29.324
protocol, so you don't have to write anything down or remember them.

295
00:18:29.384 --> 00:18:31.524
And there's two ways to export data.

296
00:18:31.744 --> 00:18:35.374
The first is in this app, and the second is in the data app that we have.

297
00:18:36.084 --> 00:18:39.124
So if you hit this export button, you can see the items you can

298
00:18:39.164 --> 00:18:41.644
export. So maybe I just want the CSV.

299
00:18:42.384 --> 00:18:45.564
And then once I press next, there's the different destinations.

300
00:18:45.624 --> 00:18:49.564
So we do have USB, or you can export via email, network

301
00:18:49.644 --> 00:18:51.164
folder, or network printer.

302
00:18:53.484 --> 00:18:55.884
Now, I do want to show you the data app.

303
00:18:57.104 --> 00:19:01.004
So as you lift the arm and you wipe away, your data is automatically

304
00:19:01.024 --> 00:19:01.404
stored.

305
00:19:02.244 --> 00:19:04.764
So once you open the data app, you'll select by app.

306
00:19:05.504 --> 00:19:09.444
So I'm going to go into AO/PI. You can select by a certain protocol if you

307
00:19:09.524 --> 00:19:13.444
work with one protocol, or you could recall a week's worth of date

308
00:19:13.524 --> 00:19:14.084
if need be.

309
00:19:15.284 --> 00:19:19.134
So I'm just going to take this count, add it to a report, because I don't want

310
00:19:19.184 --> 00:19:19.964
all of these counts.

311
00:19:21.904 --> 00:19:25.114
And then I'm going to click here, and if I were to press that export button, it

312
00:19:25.124 --> 00:19:27.504
would be the same way as if I were in the app.

313
00:19:27.964 --> 00:19:31.764
Another cool feature that we have, if you press on results list and go to

314
00:19:31.784 --> 00:19:32.194
graphs,

315
00:19:33.004 --> 00:19:36.004
we have a lot of different graphs that we show here on screen.

316
00:19:37.824 --> 00:19:41.734
So now that I'm done with the demo, I do want to talk a little bit more about

317
00:19:41.784 --> 00:19:43.184
the CellDrop itself.

318
00:19:48.424 --> 00:19:52.104
So we do have four models. We have a fluorescence and brightfield

319
00:19:52.164 --> 00:19:54.084
model, and a brightfield-only model.

320
00:19:54.564 --> 00:19:58.224
We have a 4X objective that can count cells between four and

321
00:19:58.424 --> 00:20:02.364
400 microns, and a 10X objective that can count between two

322
00:20:02.374 --> 00:20:03.424
and 20 microns.

323
00:20:06.124 --> 00:20:09.224
And we can support a wide variety of applications such as

324
00:20:09.244 --> 00:20:12.344
protoplasts, stem cells, algae, and even yeast.

325
00:20:17.424 --> 00:20:21.324
And along with our regular counting apps, we've also developed two

326
00:20:21.364 --> 00:20:25.124
machine learning apps. So we have an organoids app and a hepatocytes app.

327
00:20:25.464 --> 00:20:29.214
So the organoids app was designed for organoid, spheroid, or tumor

328
00:20:29.284 --> 00:20:32.724
sphere cell types, and this app reports counts, size

329
00:20:32.784 --> 00:20:36.724
distributions, and density. And then the hepatocytes app is

330
00:20:36.764 --> 00:20:40.014
great for drug metabolism and toxicology workflows.

331
00:20:40.454 --> 00:20:43.813
So this app reports live and dead hepatocytes along with

332
00:20:43.824 --> 00:20:47.784
leukocytes and debris. Now, the counting algorithms have already

333
00:20:47.804 --> 00:20:51.544
been trained by me and the rest of my colleagues, so it does not need to be

334
00:20:51.584 --> 00:20:52.784
trained on your samples.

335
00:20:58.264 --> 00:21:01.944
If you're sitting there right now and you may work with samples that are

336
00:21:01.984 --> 00:21:05.824
biohazardous and you need to use slides in your workflow, you can

337
00:21:05.864 --> 00:21:09.724
still use slides on the CellDrop. We do have a slide mode.

338
00:21:10.644 --> 00:21:14.584
You can use our Dinovix reusable slide or select

339
00:21:14.624 --> 00:21:15.563
disposable slides.

340
00:21:20.524 --> 00:21:23.784
So now that I've talked about the instrument, I want to take this time to thank

341
00:21:23.844 --> 00:21:27.674
everyone who has not only bought our instruments, but even taken the time

342
00:21:27.724 --> 00:21:31.604
to review our instruments. So without each and every single one of you, we

343
00:21:31.644 --> 00:21:35.484
would not be able to continue to create instruments that sit at the

344
00:21:35.524 --> 00:21:37.194
forefront of cutting-edge research.

345
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To wrap up this webinar, I want you all to leave with two things in

346
00:21:45.844 --> 00:21:49.624
mind. First, that the direct pipette technology is unique

347
00:21:49.684 --> 00:21:53.564
because it reduces plastic waste, it has cost benefits, and

348
00:21:53.604 --> 00:21:57.574
it's fast and easy to use. The second thing I want you to walk away with is

349
00:21:57.604 --> 00:22:01.344
the performance of the CellDrop. So the three different chamber heights

350
00:22:01.404 --> 00:22:04.934
allow for a wider dynamic range. It is feature-rich and

351
00:22:04.944 --> 00:22:07.394
produces high quality and accurate results.

352
00:22:07.744 --> 00:22:09.444
And finally, it is fully networked.

353
00:22:11.304 --> 00:22:11.964
Thanks, Cory.

354
00:22:12.884 --> 00:22:16.824
Got a lot of questions coming. So maybe we can make a start on those

355
00:22:16.864 --> 00:22:17.304
straight away.

356
00:22:18.824 --> 00:22:22.264
Okay. So first question was that you mentioned algae.

357
00:22:23.164 --> 00:22:25.184
Can you do native fluorescence of chlorophyll?

358
00:22:26.084 --> 00:22:29.264
Yeah, that's one of the cooler applications that our customers use this instrument

359
00:22:29.364 --> 00:22:33.284
for. So we do have customers that use the CellDrop to count

360
00:22:33.344 --> 00:22:37.184
their intrinsically fluorescent chlorophyll samples, and we do have tech

361
00:22:37.224 --> 00:22:39.584
notes on our website that describe how we can do that.

362
00:22:41.964 --> 00:22:42.783
Cool. Thank you.

363
00:22:44.544 --> 00:22:45.244
Question about

364
00:22:46.084 --> 00:22:47.243
two kind of software ones.

365
00:22:48.144 --> 00:22:52.064
How many samples can you store on the instrument, and does the

366
00:22:52.104 --> 00:22:54.844
instrument allow for multiple user accounts?

367
00:22:55.824 --> 00:22:59.744
That's a great question. We do not have a definitive number of

368
00:22:59.764 --> 00:23:03.724
counts that can be stored on this instrument, just because it takes into account

369
00:23:04.504 --> 00:23:08.424
not only counts, but also images. But it does have 256 gigabytes

370
00:23:08.464 --> 00:23:11.864
of storage. And we have a users accounts app

371
00:23:12.264 --> 00:23:13.024
that can

372
00:23:13.944 --> 00:23:16.644
input a lot of users within the CellDrop.

373
00:23:18.424 --> 00:23:20.664
And it's in the thousands of samples, right?

374
00:23:21.404 --> 00:23:21.644
Mm-hmm.

375
00:23:23.824 --> 00:23:24.064
Okay.

376
00:23:25.404 --> 00:23:28.024
Can you graph multiple samples together?

377
00:23:28.104 --> 00:23:29.804
So you showed the graph questions.

378
00:23:29.904 --> 00:23:33.804
Can you overlay them and have multiple samples in those graphs?

379
00:23:34.964 --> 00:23:38.784
Yeah, you can have, for the graphs feature, you can see different samples

380
00:23:38.884 --> 00:23:39.624
side by side

381
00:23:40.504 --> 00:23:42.224
with all of the different features that we have.

382
00:23:42.284 --> 00:23:45.344
So we have live cells, dead cells only, viability,

383
00:23:46.564 --> 00:23:47.004
size.

384
00:23:49.284 --> 00:23:51.104
A couple of questions about the PBMC

385
00:23:52.124 --> 00:23:55.944
samples. The first question was which dye

386
00:23:56.024 --> 00:23:59.904
is the best for counting PBMCs? And I think it's

387
00:23:59.914 --> 00:24:02.604
probably the one that you were using, which was AO/PI, right?

388
00:24:03.264 --> 00:24:06.204
Mm-hmm. Yeah. Not trying to be biased, but yes.

389
00:24:07.284 --> 00:24:10.684
Yeah, no, I think it's reasonable. I think it's a good dye for that.

390
00:24:11.984 --> 00:24:14.504
The other question about PBMCs, came in

391
00:24:16.284 --> 00:24:20.024
asking about all of the cells in

392
00:24:20.124 --> 00:24:23.204
that particular image that weren't counted.

393
00:24:23.244 --> 00:24:26.864
There were a lot of cells in there that weren't counted, and the question was what

394
00:24:26.924 --> 00:24:29.303
were they and why weren't they counted?

395
00:24:30.516 --> 00:24:34.416
Yeah. So all the darker cells in the background that were not counted that you all

396
00:24:34.456 --> 00:24:37.726
saw were red blood cells, and that's just because they're

397
00:24:37.776 --> 00:24:41.696
non-nucleic cells, and AO/PI does not bind to those cells.

398
00:24:42.736 --> 00:24:42.896
Yep.

399
00:24:45.756 --> 00:24:47.776
So we've got a load more come in as well.

400
00:24:49.436 --> 00:24:53.156
Let me just catch up on these. There was a question about the machine

401
00:24:53.256 --> 00:24:54.416
counting of organoids.

402
00:24:56.136 --> 00:24:58.076
Is it a specific type of organoids?

403
00:24:58.116 --> 00:25:01.896
Does it depend on size, or is it trained on multiple species,

404
00:25:01.956 --> 00:25:02.695
multiple types?

405
00:25:03.736 --> 00:25:07.336
Yeah. That's a great question. So the organoids app does depend on

406
00:25:07.416 --> 00:25:10.135
size. We have two different chamber heights for that app.

407
00:25:10.336 --> 00:25:14.136
We have a 200-micron chamber height and then a 400-micron chamber

408
00:25:14.176 --> 00:25:18.116
height. I would definitely say the max organoid

409
00:25:18.156 --> 00:25:21.696
that we can count is getting up to the 400 microns in diameter.

410
00:25:21.936 --> 00:25:25.236
But if you are definitely interested in learning more about that app, we do have a

411
00:25:25.276 --> 00:25:28.036
tech note, or you can reach out to me or one of my colleagues.

412
00:25:29.076 --> 00:25:29.236
Yeah.

413
00:25:30.816 --> 00:25:31.036
Cool.

414
00:25:32.016 --> 00:25:35.616
There was a question from a current user about how you

415
00:25:35.716 --> 00:25:36.936
change the chamber height

416
00:25:37.776 --> 00:25:41.276
and if you could explain that. I don't know if we can switch back to the

417
00:25:41.316 --> 00:25:43.295
instrument or not, whether we have that set up.

418
00:25:45.616 --> 00:25:48.136
Yeah. So we could definitely show this screen again.

419
00:25:48.736 --> 00:25:52.216
So it would be in the protocols section of

420
00:25:52.276 --> 00:25:55.696
the app. So I'm just going to pick the Brightfield app,

421
00:25:57.136 --> 00:26:00.496
and then once I get onto protocols, if everybody can see my screen, I'm going to

422
00:26:00.516 --> 00:26:02.816
hit this plus button here to create a new protocol.

423
00:26:03.536 --> 00:26:06.316
And then towards the bottom left, you will see chamber height.

424
00:26:06.816 --> 00:26:10.686
So once you select one of these, maybe the 400 or the 50, and

425
00:26:10.716 --> 00:26:14.336
then you're going to have to save that protocol and then switch to that protocol

426
00:26:14.396 --> 00:26:16.476
because it will always go to the default.

427
00:26:16.836 --> 00:26:20.716
Once you do either the 400 or the 50, you'll see the arm either raise

428
00:26:20.776 --> 00:26:24.036
or lower, and that's how you know that you changed your chamber height.

429
00:26:25.336 --> 00:26:28.806
Perfect. So, good, before you get out of that... Yeah, too late.

430
00:26:29.316 --> 00:26:32.446
There was a related question that I've just seen that was

431
00:26:32.856 --> 00:26:36.566
about how you handle non-circular

432
00:26:36.596 --> 00:26:37.076
cells.

433
00:26:38.036 --> 00:26:39.455
This is probably a good screen to

434
00:26:40.496 --> 00:26:41.295
demonstrate that.

435
00:26:41.816 --> 00:26:41.976
Yeah.

436
00:26:42.876 --> 00:26:46.576
So we have an irregular cell mode. So a lot of automated cell

437
00:26:46.616 --> 00:26:50.576
counters are produced to count circular or near perfect circular cells.

438
00:26:51.096 --> 00:26:54.756
The CellDrop can get away with it a little bit, and we have an irregular cell mode.

439
00:26:55.336 --> 00:26:57.116
This information button is really good.

440
00:26:57.156 --> 00:27:01.116
It shows you two pictures of what an irregular cell mode looks like.

441
00:27:01.196 --> 00:27:04.686
And even with this a little bit funky morphology, we're still highly

442
00:27:04.776 --> 00:27:06.036
accurate.

443
00:27:06.116 --> 00:27:06.236
Mm-hmm.

444
00:27:09.416 --> 00:27:12.956
That's great. I had a couple more questions on

445
00:27:14.196 --> 00:27:17.476
cleaning and what is the required periodic

446
00:27:17.516 --> 00:27:20.516
maintenance, so between samples and periodically.

447
00:27:21.496 --> 00:27:24.156
Yeah. This instrument's really maintenance-free.

448
00:27:24.436 --> 00:27:28.296
As you saw between samples, I was wiping away with a Kimwipe, and my sample

449
00:27:28.356 --> 00:27:32.116
surface was clean. If there is anything on your sample surface

450
00:27:32.176 --> 00:27:34.556
that's left behind by any

451
00:27:35.496 --> 00:27:39.336
debris or anything like that, you can pipette in any alcohol of your

452
00:27:39.396 --> 00:27:43.376
choice or bleach just like you would if you were loading a sample

453
00:27:43.416 --> 00:27:46.596
and just wipe away, and that sample surface will be clean.

454
00:27:49.536 --> 00:27:52.966
There's a couple that I'll group together here, which are asking

455
00:27:53.056 --> 00:27:56.996
about how the CellDrop deals with

456
00:27:57.376 --> 00:27:59.716
clumping cells or aggregated cells.

457
00:28:00.216 --> 00:28:04.066
So there's one question about particularly sticky cells, how well does

458
00:28:04.096 --> 00:28:07.896
it work with them, and also, can you see

459
00:28:09.296 --> 00:28:12.746
how big the clumps are? Do you have any way of seeing

460
00:28:13.356 --> 00:28:16.606
what the cluster size of cells is?

461
00:28:17.776 --> 00:28:21.696
Yeah. So the CellDrop has a declustering algorithm that

462
00:28:21.896 --> 00:28:23.076
it's bread and butter.

463
00:28:23.936 --> 00:28:27.696
So once you count cells, this algorithm can pick out

464
00:28:28.356 --> 00:28:32.076
single cells in a cluster of cells and count those as one.

465
00:28:32.656 --> 00:28:36.496
A few apps do report cluster measurements as well.

466
00:28:39.776 --> 00:28:43.636
So it'll give you a breakdown of clusters of one, two, three,

467
00:28:43.736 --> 00:28:45.066
four, five cells?

468
00:28:45.676 --> 00:28:49.386
Correct. And you can see that in the CSV file or

469
00:28:49.456 --> 00:28:50.516
in the PDF file.

470
00:28:52.396 --> 00:28:52.656
Great.

471
00:28:54.276 --> 00:28:58.225
There's a question about using trypan blue or erythromycin B and

472
00:28:58.276 --> 00:29:02.036
whether the dilution is taken account of, so diluting it with the

473
00:29:02.876 --> 00:29:05.996
reagent itself, or do you have to do that afterwards?

474
00:29:07.156 --> 00:29:10.795
Yeah, that's a great question. So we already factor in that one-to-one

475
00:29:10.836 --> 00:29:14.656
dilution into the protocol, and that's the nice thing about you can create your

476
00:29:14.716 --> 00:29:18.696
own protocol. So if you do any external dilutions, you can

477
00:29:18.776 --> 00:29:21.936
add that into the protocol, and the instrument will do the math for you.

478
00:29:22.156 --> 00:29:24.176
So there is no need for external math.

479
00:29:28.556 --> 00:29:32.416
There's a question about optimizing protocols and whether

480
00:29:32.456 --> 00:29:33.236
there is a sort of

481
00:29:34.436 --> 00:29:37.276
stepwise process to go through

482
00:29:38.716 --> 00:29:40.996
to optimize a protocol.

483
00:29:42.316 --> 00:29:43.356
Yeah. So each app

484
00:29:45.116 --> 00:29:49.036
has its own protocol settings that you can optimize, but I'm going to say the

485
00:29:49.076 --> 00:29:52.696
easiest thing to do is to reach out to me or one of my team members.

486
00:29:53.136 --> 00:29:57.116
We can easily optimize a protocol for your cell type, and then we

487
00:29:57.156 --> 00:30:00.756
send that protocol back to you so you can input it on your instrument, and you

488
00:30:00.796 --> 00:30:04.376
don't have to worry about playing with the settings and making sure it's working

489
00:30:04.396 --> 00:30:04.876
properly.

490
00:30:06.976 --> 00:30:07.336
Yeah.

491
00:30:08.636 --> 00:30:11.956
And it's fair to say, I think, in my experience, that the default protocols get you

492
00:30:12.016 --> 00:30:15.956
a long way, right? So the default protocols are really well-optimized

493
00:30:15.996 --> 00:30:18.396
for the majority of cell types that we use.

494
00:30:19.376 --> 00:30:21.376
And then for anything else, we kind of have the

495
00:30:22.836 --> 00:30:26.696
options in the software that you can use to finesse optimization.

496
00:30:27.436 --> 00:30:31.396
Do you have a way to update the apps on the instrument

497
00:30:31.556 --> 00:30:32.156
over time?

498
00:30:33.088 --> 00:30:35.948
Yes. So all of our software updates are free.

499
00:30:36.168 --> 00:30:39.938
They come out every six months or so, and you can

500
00:30:39.988 --> 00:30:42.078
update the instrument in two different ways.

501
00:30:42.108 --> 00:30:45.888
First is downloading the zip file from our website onto your USB,

502
00:30:46.368 --> 00:30:50.268
or if you're able to connect the instrument to their network via Wi-Fi or

503
00:30:50.308 --> 00:30:52.448
ethernet, you can download the update through there.

504
00:30:55.848 --> 00:30:56.088
Great.

505
00:30:57.068 --> 00:31:00.068
We also have a question about whether the media affects the

506
00:31:00.208 --> 00:31:02.428
AO/PI stain.

507
00:31:03.768 --> 00:31:05.308
Yeah, that's a really good question.

508
00:31:06.308 --> 00:31:10.228
The only media as of right now that can probably have a little

509
00:31:10.288 --> 00:31:14.088
bit of a problem is RPMI media, and that's just because of

510
00:31:14.128 --> 00:31:17.548
phenol red. But there are some workarounds for that.

511
00:31:17.868 --> 00:31:21.728
You can dilute your samples into PBS, and

512
00:31:21.738 --> 00:31:23.708
your AO/PI signature will still be strong.

513
00:31:25.268 --> 00:31:29.008
Perfect. And I think the final question that I can probably take

514
00:31:29.128 --> 00:31:32.768
here is about calibration and whether it

515
00:31:32.808 --> 00:31:34.668
needs an annual calibration.

516
00:31:35.648 --> 00:31:39.398
That's a great question. So this instrument is calibrated for life just

517
00:31:39.428 --> 00:31:43.248
because there is an initialization process that happens each time that you

518
00:31:43.288 --> 00:31:46.668
turn the instrument on. That process just makes sure the

519
00:31:46.728 --> 00:31:50.408
software and hardware are working together and communicating properly.

520
00:31:53.008 --> 00:31:54.268
Perfect. Thank you, Corey.

521
00:31:55.088 --> 00:31:58.608
There are other questions. So some of them are very

522
00:31:58.728 --> 00:32:00.508
specific to different cell types.

523
00:32:01.548 --> 00:32:05.328
So what I might do is say that we'll wrap up the Q&A there.

524
00:32:05.348 --> 00:32:08.988
If we haven't got to your question, I promise we will absolutely follow up with

525
00:32:09.048 --> 00:32:12.748
you. Either Corey or one of the apps team will definitely

526
00:32:12.788 --> 00:32:13.808
answer your question

527
00:32:15.068 --> 00:32:15.568
for you.

528
00:32:16.668 --> 00:32:20.508
After the webinar, we will send you an email with a link to a recording as well.

529
00:32:20.948 --> 00:32:22.628
So if you want to share it with your friends or

530
00:32:23.488 --> 00:32:26.248
look back at any of the particular details, it'll be there.

531
00:32:27.128 --> 00:32:28.828
We'll also put a little survey in there.

532
00:32:29.808 --> 00:32:33.068
We'd really appreciate it if you fill in the survey because it helps us to develop

533
00:32:33.128 --> 00:32:35.288
content that works better for you.

534
00:32:36.548 --> 00:32:40.068
If you have any questions, please feel free to get in touch with us.

535
00:32:40.108 --> 00:32:43.888
All of our details are on deNovix.com, of course.

536
00:32:43.968 --> 00:32:47.468
And if you're interested in the new Squid Pipette, all of the details are on the

537
00:32:47.508 --> 00:32:48.498
website as well.

538
00:32:49.848 --> 00:32:50.128
So,

539
00:32:51.628 --> 00:32:55.128
I would just like to say thank you again, Corey, and thank you to

540
00:32:55.188 --> 00:32:58.368
everyone attending today for a lot of great questions.

541
00:32:58.548 --> 00:32:59.528
Really appreciate it.