| | 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,
Last edited by Warthaug; 10-08-2009 at 01:15 PM.