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Percoll for PMN-Isolation

Percoll for PMN-Isolation - Protocols and Methods Forum

Percoll for PMN-Isolation - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 10-06-2009, 08:03 AM
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Default Percoll for PMN-Isolation



Dear all,

I am working on Neutrophil-Chemotaxis. Up to know I jused an isolation protocoll based on Ficoll. As Ficoll might diminish chemotactic activity, I would like to try Percoll. Unfortunately I got some problem with the protocols I found, some do ultra-centrifugation for gradient preparation, others don' t?! Is there a way to avoid ultra-centrifugation?

Thanks for your help

BJ
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Old 10-08-2009, 01:13 PM
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Default Re: Percoll for PMN-Isolation

I spent my whole PhD isolating neutrophils & preforming chemotaxis assays on them. First question, what source are you using for your neuts? If you're using blood than ficol-hostopaque 1077 is exactly what you need. If you're trying to get neuts from mouse or rats, its not really possible to get enough blood without using a lot of animals, so bone marrow is a better source (and requires percol).

I'll post both procedure below, blood followed by bone marrow. Just a few pieces of
advice in regards to both:

1) Always ensure the centrifuge is balanced and does not vibrate. Even small vibrations will activate neutrophils.
2) With the exception of the dextran step for the blood-based isolation, keep the cells cold and in calcium-free media. This will reduce the chance of activating the cells.
3) Anywhere I wrote HBSS you can supplement the media of your choice. To ensure proper function the media should have calcium and magnesium in it.
4) DO NOT USE EDTA/EGTA as an anti-coagulent. This results in a poor isolation and often screws up the cells. Use ACD as an anticoagulent instead (250ml dH2O, 7.36g citric acid, 14.71g sodium citrate, 9.91g dextrose)
5) Both procedures require nothing more than a bench-top microfuge and a standard swinging-bucket centrifuge - there is no need for ultracentrifugation.

From blood (human, cat, dog, pig, etc) - isolates 2-8x10^7 neutrophils
1. Pipet 4 ml of ACD into a 50 ml conical tube and pour 20 ml of whole blood down the side of the tube. Gently invert the tube several times to mix.
2. Pipet 12 ml of 6% Dextran / 0.9% NaCl solution into the ACD/blood mixture and invert 18-20 times to ensure adequate mixing. Let the tube stand at room temperature for 45min - 1hr, or until separation is complete.

note: dextran should be 150,000MW

3. Turn on the centrifuge to 4C.
4. Return to the settling blood and pipet the yellowish supernatant into a 50 ml tube, and top with PBS. Spin at 1150 rpm (200x g) for 12 minutes at 4C using low brake.
5. Suspend neutrophils in 12ml of ice-cold ddH2O for 20 seconds. Stop lysis reaction by adding 4ml of 0.6M KCl. Bring volume up to 50ml with PBS. Spin at 1300RPM (250x g) for 6 minutes at 4C using low brake. Repeat if necessary.
6. Discard the supernatant, and resuspend the pellet in 2 ml of PBS.
7. Layer the cell suspension over 2 ml of Ficoll-Hypaque (Sigma 1077) in a 15 ml tube. Spin at 1500 rpm (365x g) for 30 minutes at 4C with no brake.
8. When the cells have finished spinning suck off the supernatant, using a transfer pipette. Resuspend the pellet in 1 ml HBSS, transfer to a new tube and bring the volume up to 2ml HBSS.
9. Count & dilute the cells to the desired concentration

From bone marrow - isolates ~0.5x10^7 per mouse
1)Anesthetise mice (note steps 1&2 can be skipped if whole blood or serum is not required).
2)Collect blood via cardiac puncture, collect in a hepranized syringe. Spin blood 1750RPM, 15 minutes, RT, and collect serum. Dilute serum to 10% in fresh HBSS.
3)Sacrifice mice using cervical dislocation. Isolate tibia’s and femurs, remove muscle.
4)Remove ends of bone and flush out marrow using ~2.5ml PBS and a 25 gauge needle, collect in a 50ml tube.
5)Break-up marrow using a 19 gauge needle and syringe. Bring volume to 50ml with PBS.
6)Pellet cells at 1300RPM, 4oC, 6 minutes.
7)Make a 3x 2ml discontinuous percol gradient consisting of 72, 64 and 52% percol in PBS respectively.
8)Suspend neutrophil pellet in 2ml of 52% percol. Layer 2ml of 64% followed by 2ml of 72% percol below the 52%/neutrophil layer. Spin 2600RMP, 4oC for 30 min.

note: In this case, the % assumes a stock solution (i.e. 100%) of 10ml 10X PBS plus 90ml percol

9)Neutrophils will form a band between the 64% and 72% layers, collect this band in a 50ml tube and suspend it in PBS.
10)Spin down cells at 1500RPM, 6 minutes. Resuspend pellet in 0.25ml HBSS + 10% mouse serum.
11)Count cells, dilute to desired concentration.

Hope that helps,

Bryan

Last edited by Warthaug; 10-08-2009 at 01:15 PM.
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Old 10-08-2009, 04:26 PM
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Default Re: Percoll for PMN-Isolation

Dear Bryan,

thanks very much for your reply. I am using PMN isolated from human whole blood. I was thinking about Ficoll as well, but apparantly both Ficoll and Dextran seem to impair neutrophil motility (Sroka et al, EUR J Cell Biol 2009). I just did a run with Percoll @ 1,077 g/ml (or say, it should be that...). I got quite a lot of PMN, but unfortunately also some lymphocytes...
What kind of chemotaxis assays did you use in the past? Did you get good PMN motility with Ficoll?

Regards

Björn
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Old 10-09-2009, 01:25 PM
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Default Re: Percoll for PMN-Isolation

I did under agarose chemotaxis assays, ibidi chemotaxis chambers, transwells and zigmond chambers, plus a few of my own design. My isolation methods worked well in all cases, and we never saw the chemotaxis defects they claim to see.

We also did a lot of chemotaxis assays in animals (i.e. watching neutrophils crawl around in blood vessels and tissue), and the chemotaxis we observed in vitro was nearly identical in terms of speed and directionality to that observed in vivo. Given that our in vitro model systems perfectly replicated what we observed in vivo I am quite confident the isolation methods are fine. A few points in "rebuttal" to the paper:

Firstly, they used low-molecular weight dextran and ficol compared to what I use. It was established in the 1960's that the low-molecular weight variants of these products impair neutrophil function. Basically, Sroka et al set up these experiments to fail, or at least were completely unware of the 50-ish years of literature surrounding neutrophil isolations. You'll note that my protocol calls for 150,000MW dextran and ficol 1077; both much larger molecules than the 50,000MW detran/ficol 400 they are using.

Secondly, the movement and spreading of unstimulated neutrophils is a sign of an activated prep. In our hands we treated this pre-experiment activation as grounds for throwing away the prep. In contrast, Sroka et al use this as a sign of the prep being good - once again, the literature from the 1960's shows that neutrophils in blood do not spread of migrate randomly, which they (and I) took as a sign that a neutrophils from a proper isolation technique also should no engage in these activities.

Bryan
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Old 10-14-2009, 06:24 PM
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Default Re: Percoll for PMN-Isolation

Dear Bryan,

I wasn´t aware of the differences in the Ficoll preparations! Thanks for your informations, I think I´ll run a couple of assays with both methods...

Regards

Björn
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Old 10-14-2009, 07:16 PM
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Default Re: Percoll for PMN-Isolation

Its not widely advertised that ficol comes in different sizes. Ficol - like dextran - is a polymer of sugars. Differing degrees of polymerization give differing sized molecules, and different sizes act in vastly different ways.

For reasons I do not understand, smaller sized ficol or dextran seem to affect neutrophils in a negative way. Likewise, excessively large dextran also affects neutrophils - mega-dalton sized dextran is phagocytosed!

In either case you need to hit the sweet spot - 150,000MW for dextran, and 1077 for ficoll.

Bryan
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Old 11-10-2009, 04:08 PM
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Default Re: Percoll for PMN-Isolation

Dear Bryan,

I used Ficoll for isolation and my chemotaxis results are pretty good! :-) Just one thing that I do not understand: after lysis of RBC with NH4Cl I layer the cell suspension on Ficoll and centrifuge for 30min. I do all these stepts including centrifugation at 4*C. From time to time, I do not have any pellet at all after centrifugation?!?! The Sigma-guys recommended me to do all at room temperature as 4*C may cause clumping of my cells, but I would like to avoid room temperature if possible. Also, there are times that I can isolate lots of PMN with the same procedure?! I don´t get it...

Regards

Björn
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Old 11-11-2009, 02:21 PM
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Default Re: Percoll for PMN-Isolation

I have no experience using NH4CL for RBC lysis, and I suspect that is where your problem lies.

As I mentioned in the above protocol I always used ddH2O for the lysis, and then re-stabilised the WBCs by adding concentrated KCl. This always worked excellently for us. I would recommend trying that. The key is being gentle:

1) Suspend the cells rapidly, by squirting the pellet directly with the ddH2O, then suck the pellet into the pipette and expel it once to finish breaking up the pellet. Avoid blowing bubbles through the solution

2) Let sit 20sec (start your count as soon as you add the ddH2O.

3) Quickly, but gently, add the KCl.

You can do this isolation at RT, or even 37C. But this greatly increases the risk of activating the cells during the isolation procedure.

Bryan
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Old 05-17-2013, 05:26 PM
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Default Re: Percoll for PMN-Isolation

Hi! I am working on neutrophil function and am trying since last 2 months to isolate neutrophils from mice bone marrow using 62% percoll. BUt the problem is I never get a neutrophil pellet. I get at the best scenario two band at the interface separated by a very narrow difference which makes it very difficult to extract the band separately..most of the times i dnt even get two bands.A brief outline of my protocol..
After getting the bones, Flush out the bone marrow with HBSS ( without Ca or Mg) prep( .5% FBS +20mM HEPES),
Sucking out the flushed out marrow through 18g needle to break the aggragates.
Spin down cells at 400g for 5 min
resuspend cell pellet in 0.2% NaCl (RBC lysis) for 45 seconds.
Restore osmolarity by adding 14 ml 1.2% NaCl
strain throught cell strainer
Spin down (400g, 5min)
resuspend in HBSS prep and layover very gently on 5ml 62% percoll( prepared in HBSS prep)

After this step I am always confused since as i said earlier that i never get a pellet..but i try to collect the lower band of cells and wash them twice in HBSS prep and use the cells i get. Cytospin have shown good slide with very few monocytes but flow cytometry results show all contaminations with monocytic lineage..PLease help
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Old 05-17-2013, 05:32 PM
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Default Re: Percoll for PMN-Isolation

I forgot to add that all the steps are performed at 25 Degree C and percoll centrifugation is done at 1000g for 30 min
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