PMN Neutrophil Cell Isolation

[kiki06]

Hello there,
I would like to isolate polymorpho neutrophil cells and culture them.

Does anyone have any tips or a protocol to help?

Thank you all
Kiki

[dnamolecule]

Hello kiki!

here is a protocol modified from the posted one by Bryan Heit:


Neutrophil isolation is easy, however they are extremely sensitive cells.

This protocol can be used with any mammalian blood (has been performed on humans, mice and cats).

Tips for Neutrophil Isolation:

• Isolate and complete all steps at 4C as this will help to limit activation
• Avoid jarring vibrating the isolation tubes, as this can activate the cells
• Do NOT use glass - the cells will stick on the glass and not come off

NEUTROPHIL ISOLATION PROTOCOL

The protocol is a three step purification protocol of neutrophils:

STEP 1) Dextran sedimentation - this removes most of the RBC's. The RBC's sediment to the bottom of the tube, while the leukocytes and lymphocytes remain suspended in solution.

STEP 2) Hypotonic lysis - this removes any remaining RBC's and platlets.

STEP 3) Ficol sedimetntation - this separates mononuclear cells from neutophils. The neutrophils sink to the bottom of the ficol, mononuclear cells remain at the ficoll/isoalte interface.

You can achieve 98% pure neutrophils or better using this protocol, with little to no activation.

Steps in Detail:


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. (for other volumes use 1ml ACD for every 5ml blood).

2. Pipet 12 ml (50% of blood volume) of 6% Dextran / 0.9% NaCl solution into the ACD/blood mixture and invert 18-20 times to ensure adequate mixing. Pipette the mixture into 4 15ml tubes (~10ml/tube). Let the four tubes stand at room temperature for 45min - 1hr, or until separation is complete.

3. Return to the settling blood and pipet the yellowish supernatant into a 50 ml tube. Spin at 1150 rpm for 12 minutes at 4C using a low brake.

4. Discard the supernatant and resuspend in 12 ml of the ice-cold ddH2O. Suck up and dispense repeatedly to break the pellet. After 20 seconds add add 4 ml of 0.6 M Kcl and mix several times. Dilute the solution to 50 ml with PBS. Spin at 1300 rpm for 6 minutes at 4C using a high brake.

5. Repeat Step 4 1-2 times, until no RBC's remain.

6. Discard the supernatant, and resuspend the pellet in 2.5 ml of PBS.

7. Layer the cell suspension over 3 ml of Ficoll-Hypaque (Sigma 1077) in a 15 ml tube. Spin at 1500 rpm for 30 minutes at 4C using a low brake.

8. When the cells have finished spinning suck off the supernatant, using a transfer pipet. Resuspend the pellet in 2 ml HBSS.

9. Determine the cell conentraiton using a hemocytometer, or other device.

Cells are noe ready for use. Neutrophils will remain viable suspended in HBSS for about 4 hours at 4C, and about half that at room temperature. They can also be resuspended in RPMI instead of HBSS, although this has been known to activate them.

Here's how to make the solutions used in the protocol: ___________________________ ACD: To 250ml ddH2O add:

7. 36 gm Citric Acid

14. 71 gm Sodium Citrate

9. 91 gm Dextrose

Store at 4C ___________________ 6% Dextran: To 250ml ddH2O add:

15. 00 gm Dextran (at least 100 000 MW)

2. 25 gm NaCl

Store at 4?C _______________________________

0. 6M Kcl: To 250ml ddH2O add:

11. 18 gm Kcl

Store at 4C _____________________________



Modified from Bryan Heit's original protocol

[liono]

Hello kiki!
Hopes this help you:

Neutrophil Isolation

Neutrophils were isolated from venous blood of healthy volunteers by dextran sedimentation of erythrocytes and density-gradient centrifugation of leukocytes. All procedures up to and including density-gradient centrifugation were conducted at 22° C unless stated otherwise.

1. Blood was anticoagulated with acid-citrate-dextrose and centrifuged at 400 × g for 20 min to remove platelets.
2. The platelet-rich plasma supernatant was removed and centrifuged at 2,000 × g for 30 min to prepare platelet-poor plasma (PPP).
3. The platelet-depleted cell precipitate from the first centrifugation step was mixed with phosphate-buffered saline (PBS) (1:4). Dextran (Baxter, Warrington, UK) was added to a final concentration of 2% and erythrocytes were sedimented under gravity for 45 min at 15° C. The erythrocyte-depleted supernatant containing leukocytes was centrifuged at 400 × g for 10 min.
4. The resulting leukocyte-enriched pellet was resuspended in PPP. The leukocytes were then layered over a discontinuous gradient of Percoll (4 ml of 61.5% and 4 ml of 76%) in a 15-ml polypropylene centrifugation tube.
5. The Percoll densities were created from pure Percoll diluted with 10× PBS and PPP. Final percentages of PPP (vol/vol) in the Percoll solutions were 42.89% and 23. 97% for the 61.5% Percoll layer and 76.0% layer, respectively.
6. The Percoll densities and layered cells were then centrifuged at 400 × g for 20 min. After centrifugation, mononuclear cells were found at the plasma-61.5% Percoll layer interface, and neutrophils were found at the 61.5-76.0% Percoll layer interface.
7. Neutrophils were removed and washed twice in RPMI-1640. Cells were counted with a hemocytometer and resuspended at 106 cells/ml.
8. Cells were plated with the compounds investigated in the study in RPMI-1640 containing 10% autologous-plasma-derived serum (PDS). PDS was obtained from nonanticoagulated cell-free plasma.
9. Cell-free plasma was obtained by centrifugation of whole blood immediately after venipuncture.
10. The cell-free plasma was incubated at 37° C and PDS was derived by removal of the protein clot.
11. The purity of neutrophils was consistently greater than 99%.

Protocol from original of Sam Coward