To enrich for circulating nucleated cells from blood. The microfluidic device is first primed with one XPBS to rid of bubbles, deionized water, and 2%paraldehyde in two XPBS are then run through the device via syringe pumps prior to loading of the blood sample. Blood is then processed through the microfluidic device and collected post-processing of the sample includes centrifugation and removal of supinate.
Finally, the sample is analyzed via flow cytometry to produce fax plots. Hi, I'm William White from the laboratory of Microscale Biotechnology in the Department of Bioengineering at the University of Wool. Today we'll show you a procedure for the enrichment of circulating nucleus cells by lysis of whole blood via microfluidics.
We use this procedure in our laboratory to study inflammation in various disease processes as to ensure enrichment of leukocytes and other circulating nucleated cells without loss or activation. So let's get started. To begin this procedure, press fit access tubing of slightly larger diameter than the channel access holes in the inlet and outlet holes.
Then to provide the macro to micro interface, attach a 30 gauge syringe needle to the tubing on each of the inlets. Next, fill a one milliliter syringe with one XPBS making sure to get rid of bubbles. Connect the syringe to inlet one and push the syringe gently by hand until one XPBS flows out of inlet two.
Then immediately clamp inlet two with an office binder clip. Continue pushing the syringe until one XPBS solution reaches the outlet port of the microfluidic device. Once the solution flows outta the outlet, clamp the outlet tubing with another binder clip.
Continue further pushing the one milliliter syringe until one XPBS flows out of inlet three and clamp the inlet three port with a third office binder clip. The microfluidic device is now fully primed. Start calibrating the syringe pumps with the large Harvard syringe pump, setting the diameter configuration to 22.5 millimeters and the flow rate to 600 microliters per minute.
For the small Harvard syringe pump. Set the diameter configuration to 4.61 millimeters and the flow rate to 20 microliters per minute. Next, fill one 30 milliliter syringe with deionized water by withdrawing a solution directly from the 50 milliliter conical tube.
Remember to label the syringe, fill another 30 milliliter syringe with two XPBS 2%paraform aldehyde solution. Again, withdraw directly from a conical tube and label the syringe. Now connect the water containing syringe to inlet two and the 2%para formaldehyde in two XPBS syringe to inlet three while making sure no bubbles are trapped in the tubing or in the microfluidic device.
Then remove the clamps from inlet two, inlet three, and the outlet tubing with all three syringes connected. Proceed to put the two large syringes together on the large Harvard syringe pump and set the one milliliter syringe on the small Harvard syringe pump. Place the outlet tubing in a 50 milliliter conical waste collection tube.
Now turn on the large Harvard syringe pump holding the 30 milliliter syringes and let it run for one minute. Check that there are no bubbles and no leakage in the device or in the tubing. Then turn on the small Harvard syringe pump and let it run for an additional minute here too.
Checking for bubbles and leakage. Stop the pumps after two minutes. As the microfluidic device is now ready to be used with blood samples, the blood samples to be analyzed were collected as described in the accompanying written protocol and are used within one hour of collection.
The first tube of collected blood is discarded to rid of false positive results. Remove the one milliliter syringe from the small Harvard syringe pump, then inject one XPBS from the one milliliter syringe filling the syringe needle. To avoid introduction of bubbles, fill a one milliliter syringe with 50 microliters of one XPBS without trapping any bubbles.
Then fill the syringe with 0.5 milliliters of blood. Keep the syringe vertical while filling to avoid mixing of the PBS with the blood. Next, connect the syringe containing the blood back to inlet one.
Wipe away any excess fluid and mount the syringe vertically on the small Harvard syringe pump. Be careful not to push the blood through the tubing into the device. Remove the outlet tubing from the waste tube and place it in a clean 50 milliliter centrifuge tube sitting in an ice bucket.
Now start the run switch on the large Harvard syringe pump first and let it run for one minute. Then switch on the small Harvard syringe pump. The sample will start collecting from the outlet tubing into the 50 milliliter centrifuge tube.
Let the blood sample in the one milliliter syringe travel completely through the device, which takes approximately 20 minutes. Then stop both pumps. Now that the run is complete, centrifuge the collected sample for five minutes at 350 times G at room temperature with the break off.
Remove the supernatant by placing the pipette tip at the opposite side of the white pellet. Remove as much supernatant as possible, especially red cell debris without disturbing the white pellet. Resuspend the sample in one milliliter of flow buffer containing 1%BSA and 2%para formaldehyde in one XPBS by pipetting.
Up and down, transfer the sample to a 1.5 milliliter eend orph tube micro centrifuge, the collected sample for five minutes at four times g. At room temperature, remove the supernatant as carefully as in the previous centrifugation step. Finally, add one milliliter of flow buffer vortex to resuspend the sample and proceed to analyze the sample by flow cytometry.
Now we'll show some representative results in the form of fax plots from a blood sample enriched by the microfluidic device with analysis using flow cytometry. The importance of enrichment by lysis can be seen by a fax plot where red blood cells cloud. The scatterplot produced by the microfluidic enrichment protocol is a clean scatterplot with defined nucleated cell populations found in circulation.
The red region represents lymphocytes, green represents monocytes, blue represents granulocytes, and the purple region has yet to be characterized. Failure to remove red cell debris in the protocol produces a cluster of events in the fax plot seen in the cyan region. Populations of cells can be visualized in separate fax plots.
Lymphocytes sustained with CD three and CD four. Monocytes sustained with CD 14 and granulocytes sustained with CD 66 P.We've just shown you how to set up and run a blood sample with the microfluidic blood lysis device. When doing this procedure, it's important to remember to run the blood sample immediately to not allow the PBS to mix with the blood prior to entering the device and to avoid introduction of bubbles into the channels of the device.
So that's it. Thank you for watching and good luck with your experiments.
