Name: CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV (Video) | JoVE Journal
Uploaded: 2007-10-01T13:03:52.000+00:00
Duration: 12 min 44 s
Description: Learn microfluidic capture of CD4+ T lymphocytes using a disposable PDMS chip for HIV diagnostics in resource-limited settings. This method enables precise quantification of T helper cells from whole blood.

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5 minutes

<ol>
<li>Prepare microfluidic channel with anti-CD4+ biotinlayted surface.</li>
<li>Inject 10 ml of sample blood at 5 ml/min into the microfluidic channel with syringe pump.</li>
<li>Flush out the blood at 20 ml/min for 20 min with PBS.</li>
<li>Inject diluted FACS lysing solution (diluted to 10% from stock solution with PBS) at 5ul/min for 15 min. </li>
<li>Incubate for 15 min.</li>
<li>Inject primary CD3+ antibody solution (diluted to 10% from stock solution with PBS) at 5 ml/min for 20 min. </li>
<li>Incubate for 2 hours.</li>
<li>Flush out channel with PBS at 5 ml/min for 15 min.</li>
<li>Inject 20% AF488 CD4+ antibody, 0.2% DAPI stock solution, and 10% secondary CD3+ antibody at 5 ml/min for 20 min at the same time. </li>
<li>Incubate for 20 min.</li>
<li>Flush out the channel with PBS at 5 ml/min for 15 min.</li>
<li>Count cells under fluorescent microscope: <ol>
<li>White (optical) - Black and white: cell and dust</li>
<li>UV(DAPI) - Blue: cell only</li>
<li>Blue(CD4+) - Green: T-lymphocyte and monocyte</li>
<li>Green(CD3+) - Red: T-lymphocyte only</li>
</ol></li>
</ol>

<table><tbody><tr><td>PDMS microchannel</td><td></td><td></td><td></td><td>Fabricated by SU-8 mold</td></tr><tr><td>Blood</td><td></td><td></td><td></td><td>Donated blood from normal human subject. Mass General Hospital, 10 X high density WBC or Brigham and Women&amp;rsquo;s Hospital.</td></tr><tr><td>PBS</td><td>Buffer</td><td>Invitrogen</td><td>14190</td><td>1x Dulbecco&amp;rsquo;s Phosphated Buffered Saline.</td></tr><tr><td>FACS lysing solution</td><td></td><td>BD Biosciences</td><td>349202</td><td>Diluted to 10% from stock solution with PBS.</td></tr><tr><td>Anti-CD4+ biotinlayted Ab</td><td></td><td>Beckman Coulter Inc.</td><td>IM0704</td><td>Surface prepared with Anti-CD4+ biotinlayted Ab, diluted to 10% from stock solution with PBS/BSA after mixing with 0.2mg stock and 1ml deionized water. </td></tr><tr><td>goat anti-hCD3delta</td><td>pAB</td><td>Santa Cruz Biotechnology, Inc.</td><td>sc-1128</td><td>Diluted to 10% from stock solution with PBS</td></tr><tr><td>rabbit anti-goat IgG PE</td><td></td><td>Santa Cruz Biotechnology, Inc.</td><td>sc-3755</td><td>Secondary Ab fro anti-CD3 Ab, diluted to 10% from stock solution with PBS</td></tr><tr><td>anti-human CD4+</td><td></td><td>BD Biosciences</td><td>557695</td><td>Alexa Fluor 488 labeled, diluted to 20% from stock solution with PBS. </td></tr><tr><td>DAPI</td><td></td><td>Invitrogen</td><td>D1306</td><td>4&amp;rsquo;,6-dianidino-2-phenylindole, dihydrochloride solution, diluted to 0.2% from stock solution with PBS.</td></tr></tbody></table>

cd4+ t-lymphocyte capture using a disposable microfluidic chip for hiv

CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

Hi, my name's Richard Lin and I'm a researcher here in Professor TI Mercy's group in the Harvard MIT Division of Health Sciences and Technology. The project I'm working on is to quantify and capture CD four positive and C3 positive T lymphocytes. In patient blood is to be used in resource limited settings.So right in front of me here we have a microfill device that's composed of PDMS, which is Polymethyl Sloane. The device, the channel itself, the high is 50 micrometers high and the total volume in the device is 10 microliter. So this is a extremely precise device that we're going to use to capture T helper lymphocytes.Basically the, the diagnostic benchmark for HIV patients. So the surface of the micro fluidic device has already been prepared with protein and CD four positive antibodies in the syringe. Right now we have just PBS biological buffer at pH of 7.4 and we'll now flow this into the channel to flush out the extra CD four positive antibody.At this stage, the rate does not need to be controlled because we're just trying to flush out the extra stuff and it doesn't really matter what our rate is. This, this is where the inlets of the device is going to be and this is going to be the outlets to our, to my left and to your right here. So now I will flush out the extra antibody solution and if you can notice from the outlet you're have the fluid coming out, you know this is very minute amount, but if you look closely you can see that you air fluid coming out of the channel through the tubing.So now that we've flushed out the, the extra antibody solution, we can now prepare ourself to flow in injecting the blood. So I will now unplug the, the tubing from the inlet. So now we'll open new syringe for the blood, human blood, whole blood.So it's not a patient sample, it's a healthy sample, healthy subject sample while we prepare ourself to inject in the blood into the microfluidic device. So we'll attach the syringe onto the syringe pump. Now we'll turn on and you can see the flow rates of the syringe and we've set it to be five microliter per minute.And we'll now basically prepare ourselves to inject in the blood. We will place the syringe pumper vertically because as we know, the blood cells do sediments and we will, we'll want the cells to flow into the channel. So rather have the cell sediment to the bottom, which is right near our tubing.So now everything is set up ready to go. The volume of my device is 10 microliter per 10 microliter. And the volume that we want to flow into the channel wall is also 10 microliter.So I can tell if I flow in enough blood by just looking at the color of the channel, if it's all red all filled up, then know I flow in 10 microliter and I should stop the syringe bumper. So now I'll press a start button and it'll start sometimes, sometimes it takes a while before the blood actually gets go into a channel. And this a lot is due to like the pressure.For example, when we put the syringe on there, sometimes we would, you know, withdraw or inject the, the fluid accordingly to adjust the blood in the tubing. So it may something, it may take a while, but once it starts pumping into a channel, the flow rate is set at ly per minute, which is what we want. So we might be a little patient here and wait, sometimes you have to wait for like two minutes, three minutes before the blood start going to channel.Actually you can see right now the blood is, is starting to go in the channel. So you can tell right now the device almost completely filled up. Okay, so now we will switch the tubing back to PBS to flush out the blood that's in the channel.And to do so we will repeat what we already did to remove the tubing. Since we still need to control the rate of the flowing of the PBS, we still need the syringe pumper. However, because we're not flowing blood anymore, we don't need to leave the syringe pumper in a vertical position because there's no cells in the PBS.So now we're all set and we will now flush out the blood from the device and I will press the sharp button again right here. So as you can see, the device is kind of starting to clear out. The edges is still a little bit of blood, but it is slowly disappearing.So once all the blood is gone, we'll increase the rate from five microliter per minute to 20 microliter per minute. And the reason for this change is because in the blood there are actually two cell types that have C four positive antigens. One is what we were looking for, the T helper lymphocytes.The other one is what we call the monocytes. We wanna make sure that our device specifically capture just the T helper lymphocytes rather than both of the T helper lymphocytes and the monocytes. And the, the method that we do that is to increase the flow rate because we know that monocytes are larger in size than T helper cells.So by increasing the flow rates, the larger monocytes will experience more pressure to leave the surface than the CD four positive T helper cells. So now it's in rough of 20 minutes. So in my hand right now is, it's a solution.It's called fax licensing solution. The purpose of it for this is to ly red blood cells. So wherever red blood cells are remained in the channel, this solution will ly it.This is diluted 10, 10 times from the stock solution. So now what we're going to do is stop the syringe pumper, remove the tubing, and basically replace the solution with the one from this syringe. So I'm now stop the machine, remove the tubing, Take this off, Take it with this syringe, replace it with a new one.Okay, so now that I have everything set up, we will decrease the rate back to five microliter per minute. So we'll click the rate from 20, we'll decrease that to five and now we'll flow in the fax license solution for 20 minutes. And we'll now press start.So in front of us right now is an optical image of the device taken at 10 x magnification. So you can tell all these white dots here ourselves. So how can we make sure that these cells are the ones that we're looking for?So we use a couple different stains to make sure the first stain we use is what we call dpi, all it, it stains in DPI stains in nucleus. So basically any cells with a nucleus such as while white blood cells with all stain while say the red blood cells which don't have nucleus would not be stained and you can tell that it's all across the image. They're all these blue dots and every single one with blue.is a cell. So now the next step is to check if the cells have what we call the C four positive antigen. This is extremely important because the whole purpose for our device is to capture C four positive cells.So the next, the fluorescent antibody we use of course is C four specific. And go to the next slide. So in this picture, the, the cells ch out much dimmer is because the, the fluorescent antibody we use is usually for facts.So dilution is much higher. But if you look fairly closely, you can still tell that there are still dots that litter around this field view. They are fairly dim.But upon closer examination we can tell that these cells are in DC for positive, which is good because that's exactly what we want. And the next test is, like I mentioned before, there are two different types of C four positive cells. One are the ones that we want the T helper cells and the other are what we call the monocytes.So we again need another fluorescent antibody to differentiate between the two cells. So we know that in T helper lymphocytes they have what we call CD three positive antigens on them. While for monocytes they do not.So the next antibody we use is CD three specific, which we'll see in this picture. So again, you can tell that their dots litter around the device, they, the dots look bigger because sometimes some cells just shine up brighter, some don't as well. I mean this, a lot of stuff are up to chance in this case, but in most case, these dots in bright red dots are all cells.So we just, again, what we want because one, make sure that our cells are CD four positive and CD three positive. So in this case you can tell a, a lot of our cells are in d CD three positive, which is good. It gives us specificity in our capture.The big dos, it could be junk, but a lot of times what happens is sometimes a cell just happened to gather more CD three flushes, CD three onto themselves. So in this picture, this is one of the datas that we've collected for this micro fluid device and it's basically a cell capture pro. So in the previous one, you know, slides, whatnot, you can tell that there were a lot, you know, pictures with cells on them.But what do they really accumulate to? Well, they accumulate to a cell capture profile, in which case we can plot in the X axis we have the distance from the inlet and the Y axis we have the capture cell disc. And you can tell that throughout the device, you know, your, your density initially is extremely high and then slowly decreases, which is what we, we would expect because we're capturing the C four positive and three po, C3 positive cells from the bloodstream and they'll be de decreasing concentration of those cells in the blood sample.And this is basically how our data is usually presented.

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Learn microfluidic capture of CD4+ T lymphocytes using a disposable PDMS chip for HIV diagnostics in resource-limited settings. This method enables precise quantification of T helper cells from whole blood.

CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

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