Isolation of Neutrophils from Peripheral Blood

Materials and Reagents

1. 15 ml polypropylene tubes (Ucallm)

2. 50 ml polypropylene tubes (Ucallm)

3. 5 ml centrifuge tubes(Ucallm)

4. 2 μm syringe filter(Ucallm)

5. Transfer pipette 3.5 ml (Ucallm)

6. BD Plastipak Eccentric Luer-Slip Syringe (Fisher Scientific)

7. Serological pipettes (Ucallm)

8. Tri-sodium Citrate BP 3.8% w/v (Martindale Pharmaceuticals)

9. Dextran T500 (Pharmacosmos)

10. 9% Saline (sodium chloride) (Baxter Healthcare Corporation)

11. Percoll (GE Healthcare)

12. RPMI 1640 media (Lonza)

13. Hank’s Balanced Salt Solution (Gibco, Life Technologies)

14. Fetal Bovine Serum (FBS) (Sigma-Aldrich)

15. Penicillin-streptomycin (Gibco, Life Technologies)

16. Ethanol absolute (Fisher Scientific)

17. 6% Dextran solution (see Recipes)

18. 90% Percoll (see Recipes)

19. 70% Ethanol solution (see Recipes)

20. Media (see Recipes)

Recipes

1. 6% Dextran solution

3 g Dextran

50 ml saline

Dissolve at 37 °C in water bath and filter sterilize using a 50 ml syringe and 0.2 μm syringe filter. Once made, solution can be stored at 2-8 °C for up to two weeks

2. 90% Percoll

9 ml Percoll stock

1 ml sterile saline

3. 70% ethanol solution

Prepare 70% v/v ethanol solution with distilled water

4. Media

500 ml RPMI 1640

10% Fetal Calf Serum (Heat-inactivated)

1% Penicillin-Streptomycin

Keep the reconstituted media at 4 °C, away from light

Equipment

1. BD Vacutainer Safety-Lok blood collection set (Becton, Dickinson and Company, catalog number: 367282)

2.Water bath (NE1-8, Nickel-Electro Ltd, Weston-super-Mare, UK)

3.Class II Microbiological Safety cabinet (Walker Safety Cabinets, catalog number:50-600-467)

4.Multi-channel pipette (Alpha Laboratories, catalog number: AP80300)

5.Heraeus Biofuge Fresco (Thermo Fisher Scientific, catalog number: 75005521)

6.Large capacity centrifuge (DBJ Labcare Ltd., model: MSE Mistral 3000i)

7.Cell counting chamber (VWR, catalog number: 630-1510)

Procedure

Aside from the blood collection and centrifugation steps, carry out all work inside a Class II Biological Safety Cabinet.

A. Preparation

1.Prepare a Class II Biological Safety Cabinet for use by cleaning with 70% ethanol or similar.

2.Pre-warm the 6% Dextran solutionin a water bath set to 37 °C.

3.Place the 90% Percoll solutioninto the cabinet to allow it to reach room temperature.

4.Prepare a50 ml tube for blood collection by adding 5 ml 3.8% tri-sodium citrate as an anti-coagulant.

Note: Concentration of tri-sodium citrate = 11% total volume.

B. Blood collection

1.Obtain peripheral whole blood samples by venepuncture using a BD  Vacutainer Safety-Lok blood collection set, collecting 40 ml of blood into a 50 ml syringe.

2.Immediately transfer the blood into the 50 ml tube containing tri-sodium  citrate, running the blood slowly down the side of the tube to avoid the formation of bubbles. Invert slowly several times to mix.

C. Neutrophil isolation

1.Centrifuge the blood at 323 x g at 20 °C for 20 min. This results in the separation of the blood sample into two distinct layers of platelet-rich plasma (PRP) and blood cells.

2.Using a serological pipettefollowed by a Pasteur pipette, transfer the PRP to a  fresh 50 ml tube. Avoid disturbing the interface.

3.Centrifuge the PRP at 896x g for 20 min at 20 °C to pellet the platelets.  Following centrifugation of the PRP, transfer the supernatant (PPP) to a fresh 50 ml tube. The platelet pellet can be discarded.

4.During this centrifugation step, add 6 ml of the pre-warmed 6% dextran solution to the remaining blood cell layer from Step C1, and top up to 50 ml with saline. Replace the lid and mix gently by inversion. Take off the lid and use a Pasteur pipette to remove any bubbles that have formed around the top of the tube or in the lid.

5.Loosely replace the lid, and leave undisturbed at room temperature for 20-30min. A clear interface should be visible.

6.Following dextran sedimentation, transfer the pale upperleukocyte layer to a clean 50 ml tube using a serological pipette followed by a Pasteur pipette. Take care to not to disturb the interface.

7.Centrifuge at 224 x g at 20 °C for 6 min.

8.During the centrifugation step, the discontinuous plasma/Percoll gradient is prepared.

a. Label two separate 15 ml tubes as “upper phase” and “lower phase”.

b. To make the lower phase, add 1.02 ml of 90% Percoll (Recipe 2) and 0.98 ml PPP. Mix carefully by pipetting up and down, avoiding bubbles.

Note: For accuracy use a P1000 pipette to make the gradient layers.

c. To make the upper phase, combine 0.84 ml 90% Percoll with 1.16 ml PPP. Mix carefully as above.

d. Using a Pasteur pipette, take up all 2 ml of the upper phase and very slowly deposit it on top of the lower phase. Hold the tube containing the lower phase at a 45° angle so the liquid slowly runs down the side of the tube and sits on top of the lower phase.

Note: Avoid the formation of bubbles.

9.Once the centrifugation (Step C7) is complete, remove and discard the supernatant, and gently resuspend the leukocyte pellet in 2 ml PPP by gently swirling the tube in a circular motion.

Note: The cell pellet is soft and extra care is required when handling.

10.Deposit all 2 ml of the resuspended leukocyte population on top of the upper phase of the gradient using the same careful technique as in Step C8d.

11.Centrifuge the tube containing the gradient at 271 x g for 11 min at 20 °C with no brake.

Note: This yields three layers of cells: remaining red blood cells at the bottom of the tube, granulocytes in the middle, and peripheral blood mononuclear cells (PBMCs) closest to the top.

12.Using a Pasteur pipette, remove the PBMC layer into a clean 50 ml tube, being sure to remove as many cells as possible as residual PBMCs may contaminate the granulocyte layer.

Note: PBMCs can be resuspended in media and used in other protocols or discarded if not required.

13.Using a Pasteur pipette, remove the granulocyte layer into a clean 50 ml tube, taking as many cells as possible but avoid disturbing the red blood cell pellet.

14.Add 10 ml PPP to the tube, top up to 40 ml with Hank’s Balanced Salt Solution (HBSS) and mix by inversion to ensure an even cell suspension.

15.Count cells using a cell counting chamber.

16.Centrifuge at 504 x g for 6 min at 20 °C.

17.Remove the supernatant and gently resuspend the cell pellet in media (Recipe 4) to a final concentration of 5 x 10⁶ cells/ml.

References

1.Herman, K. D., Rahman, A. and Prince, L. R. (2020). Isolation and High Throughput Flow Cytometric Apoptosis Assay of Human Neutrophils to Enable Compound Library Screening. Bio-protocol 10(11): e3640. DOI: 10.21769/BioProtoc.3640.

2.Rahman, A., Henry, K. M., Herman, K. D., Thompson, A. A., Isles, H. M., Tulotta, C., Sammut, D., Rougeot, J. J., Khoshaein, N., Reese, A. E., Higgins, K., Tabor, C., Sabroe, I., Zuercher, W. J., Savage, C. O., Meijer, A. H., Whyte, M. K., Dockrell, D. H., Renshaw, S. A. and Prince, L. R. (2019). Inhibition of ErbB kinase signalling promotes resolution of neutrophilic inflammation. Elife 8: e50990.

3.Haslett, C., Guthrie, L. A., Kopaniak, M. M., Johnston, R. B., Jr. and Henson, P. M. (1985). Modulation of multiple neutrophil functions by preparative methods or trace concentrations of bacterial lipopolysaccharide. Am J Pathol 119(1): 101-110.

Manufacturer’s Link

Fisher Scientific: https://www.fishersci.com

electronic Medicines Compendium (eMC): https://www.medicines.org.uk›emc

Pharmacosmos: https://pharmacosmos.com

Baxter: https://www.baxter.com

GE HealthCare: https://www.gehealthcare.com

Lonza: https://www.lonza.com

Thermo Fisher Scientific: https://www.thermofisher.com › brands › gibco

Sigma-Aldrich: https://www.sigmaaldrich.com

Becton Dickinson: https://www.bd.com › en-us

Nickel-Electro Ltd: https://nickel-electro.com › standard-lab-bath

Walker Safety Cabinets: https://walkersafetycabinets.co.uk

Alpha Laboratories: https://www.alphalabs.co.uk

Thermo Fisher Scientific: https://www.thermofisher.com

DJB Labcare: https://www.djblabcare.co.uk

VWR: https://www.vwr.com

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