I am extracting granuloytes from whole blood with polymorphprep (contains dextrin and ficoll). My protocol involves extracting the granulocytes from the density layer and lysing any RBCs with pure water. The final solution I resuspend in is 350ul of HBSS with calcium and magnesium. My initial giemsa and trypan blue assay results are good.
However, my first issue is that I seem to be an order of magnitude off. I use 20mL of blood and usually get a final count of granulocytes in the 10^6 order of magnitude, but I've heard that most people get in the 10^7 order of magnitude.
The bigger issue occurs when I let the resuspended granulocytes sit for a few minutes before I perform my experiments on them. They turn into a very sticky, slimy cell pellet that tends to jam pipette tips and get thrown out by mistake. Whatever I do, they are stubborn to resuspend. I've never seen any cells behave this way before. This may also be causing my counts to be off because most of the cells are stuck. Has anyone had these issues before and have tips to solve them?
Try lysing the RBCs with ACK buffer before running the gradient. Water is causing cell lysis and DNA leakage.
Just to clarify, our procedure runs the gradient, extracts the band and then uses pure water to lyse red blood cells. Could we use the ACK buffer in place of the water?
Pellet your whole blood. Resuspend in ACK until lysis. Adjust with 1.1X volume 10X HBSS and load gradient. If desired, you can pellet again before loading the gradient.
It means they got angry and blew themselves up. You can't undo that kind of damage. If that happens, they're lost. You need avoid whatever it is that's causing them to blow up in the first place. It could be anything. Mechanical stress. Chemical exposure. Some surface they don't like. Try changing things around, like glassware vs. plasticware, temperature, fresh buffers...
Also leave the Ca and Mg out. Those are the single most likely culprit in my view.
Thanks for the tips! I'm going to start by trying ACK buffer and leaving the Ca and Mg out.
Hi Alden, for how much time your cells are in gradients (centrifugation time)? Are you using buffer with Calcium and magnesium during the washing?, if yes this is the major reason of clumping. Hypotonic lysis of RBC is usually done for 15-30 sec only. Are you using dextran sedimentation step for removal of RBCs? If you use dextran sedimentation of RBCs and then density gradient centrifugation, it will remove most of the RBC and you will get granulocytes which has negligible RBCs contamination. I will suggest to use filtered sterile buffer. If you describe your protocol here it will be helpfull to identify the problem. For the granulocyte isolation every step is very important like time, temp, speed, volume of gradient, buffer etc.
The centrifugation time to set up the gradient is 45 minutes at 500g and 19 degrees C. We then centrifuge for 10 minutes at 400g and 19 degrees to pellet the cells during the washes.
For the second question, I used HBSS without the calcium and magnesium for the first couple of washes but suspended the cells in HBSS with calcium and magnesium for the final suspension. Then I counted the cells. I am working on a project to cryoproserve the granulocytes, so I then aliquatted a volume of the suspension into various tubes. This is when I encountered the worst of the problems.
We use a specially prepared solution called polymorphprep that combines the dextran and ficoll. Our lysis is done for 30 seconds with pure water. I then add 10x PBS to make the solution isotonic and fill with HBSS without calcium and magnesium.
The only problem which I think is your cells are in gradient for a very long time (45 minutes). when I was isolating the granulocytes, I did density gradient centrifugation for 15-20 minutes only. After that I collected the desired cells and mix with 5 volume of HBSS buffer and then wash at 200g x 5minutes. First I was doing dextran sedimentation and then density gradient centrifugation. I will suggest the washing at 200g for 5 minutes only and resuspend the pellet gently in buffer.
Thank you everyone for the help. I tried the protocol today with ACK buffer, a slower centrifugation speed for pelleting, and HBSS without calcium and magnesium. These changes worked; the pellet wasn't sticky and I got a really good yield.
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