Hi,

Please Find the following protocol.

PROCEDURE FOLLOWED IN 4 degree Celsius).

1. Load venous blood on Ficoll Paque Plus (GE Health care).

2. Remove all the layer by except RBC pellet gently.

3. Re-suspend RBC pellet (e.g. 5 ml) in 30 ml HBSS without calcium magnesium.

4. Centrifuge at 250 RCF for 10 minute (zero break and zero acceleration).

5. Remove supernatant HBSS without disturbing the pellet.

6. Perform water lysis for destroying RBCs. (Add 45 ml of autoclaved water into RBC pellet, followed by add 5 ml of 10X PBS. (This procedure should be as quick as possible).

7. Centrifuge above mixture at 350 RCF for 5 minutes.

8. Repeat this process one more time in order to completely eliminate RBS. (Water lysis should not be more than 3 times).

9. Mix the pellet gently. (Do not use pipette for mixing in any step of isolation).

10. Add 15 ml of HBSS Without Calcium and Magnesium and gently mix the pellet.

11. Centrifuge at 350 RCF for 5 minutes.

12. Remove supernatant and add RPMI1640+10% FBS in it.

13. Culture cell with density of 10^5 to 10^6 cells per milliliter.

14. Confirm it by Giemsa staining/ DAPI staining.

This is the most simplest method I regularly practiced in my Lab.

Try with this method.

Best luck!

Durgesh

@ Durgesh, How much FIcoll per ml of blood do you use? Also, for step 2, do you mean to remove all the cell layers except for the RBC pellet? And if so, does this mean that neutrophils are mixed within the RBC pellet? Also, Can I use PBS instead of HBSS? We do not have HBSS in the lab. Thank you for your response

Also, how else do u mix the pellet without using a pipette?

I am a little confused on why you would take the RBC pellet. Other protocols I have seen take blood, separate it into buffy coat, rbc, plasma, and then take the fresh buffy coat and centrifuge that to get neutrophils. So I am not sure which one to follow

Mix the blood with equal amount of RPMI1640 (without serum)**. Then load on 3/4 amount of ficoll (if you have 15ml of blood+RPMI mixture then use 7-8 ml of ficoll). remember during PBMC isolation all the chemicals should be at RT (23-25 degree). Most of the neutrophils settle with RBCs. Yes, their are few population of neutrophils present in the PBMCs. But it is very difficult to isolate it without its activation. I have never used PBS instead of HBSS. If you want to use PBS then it should be without calcium and magnesium.

Answer of your next question is:

You mix neutrophil pellet by gently tapping the tube with fingers. Do not tap on the pellet side, tap away from it.

Your ficoll gradient contains following layers in descending order:

Plasma, PBMCs, Ficoll and RBC & granulocyte pellet (> 95% neutrophils+ little eosinophil and basophil population- negligible).

RBC pellet is rich with neutrophils. It is very easy to isolate resting neutrophils just lysing the RBCs.

Please find the following attachment.

Ok. Thank you very much

hi

Hi Jada,

Did the protocol mentioned by Durgesh work? I want to use it too.

and did you use PBS instead of HPSS?

Durgesh,

Thanks for the protocol. Got pure neutrophils. 

I have used RBC lyse buffer instead of water. 

RBC Lyse buffer:

NH4Cl :0.675 gm, Trizma base: 0.92 gm, EDTA: 0.133 gm, Distilled water: 100 ml. Adjust the pH to 7.3.

Durgesh,

thanks for the protocol. Actually i have tried to isolate neutrophils using the protocol from the link that you have shared, but unfortunately i didn't get the clear band of PMNs band after centrifugation. I was using Polymorprep (AXIS-SHIELD, NORWAY) as my neutrophil isolation media and should be have two clear band of PBMCs and PMNs but i only have one clear band. Really need your suggestion pls, thanks before :)

Hi Yulianna,

Nice to read you once again. I regularly used polymorpho prep and Lympho prep and combination of both for isolation of human neutrophils. If you have both of these then follow below protocol. This will give you pure neutrophils.

1. Undiluted/diluted edta treated blood layered on equal volume of Polymorphoprep (temp. 23-25 degree celcius).

Centrifuge at 500 rcf for 35-40 min at RT(23 degree Celsius) without acceleration and break.

2. You will observed upper plasma layer followed by PBMCs, PMNs, ploymorphoprep and RBCs (from top to bottom).

If you are comfortable e in separating these two cell layers then you will directly get neutrophils population. Since I am not so good at pipetting these two layer separately. I collect these two layer, mix them and re-suspend it in HBSS (without Ca and Mg + 2mM EDTA) and layered them on lymphoprep or Ficoll. Centrifuge at 600 RCF for 30 min at RT (0 acceleration and break).

3. You will observe PBMCs layer at top and PMNs pellet at bottom.

Now it is very simple to isolate PMNs. Wash once with cold HBSS. resuspend it in RPMI 1640 + 10 % FBS.

With this method you will get most purified PMNs.

I hope this will solve your problem.

Best luck.

Durgesh

Hi again Durgesh,

it also really nice and appreciate it for always share your successful experience with us . Yes, we have both polymorpho prep and ficoll in our lab, and i will try to use these combination to get pure neutrophils. For HBSS , we only have without Ca and Mg but we don't have EDTA included , is there any effect of it? thanks again :)

Hi Yulianna,

EDTA avoid unnecessary clumping of the neutrophils. It will help you to get single cell suspension.

PMN Neutrophil Cell Isolation

A easy procedure reported in article “Zagami F.:(1993) Phagocitic index: A valid method of phagocytosis study. Agg. Med. & Chir. Vol. 11, 5, 469-478, in which I showed in material and methods the experimental way.

Cellular yield: a discontinuosus gradient of Percoll (Pharmacia) is used, starting from Percoll done isotonic dissolving in it 0.15 M NaCl (solution referred at 100%). One volume of 1:2 whole blood (EDTA-K3) diluted in NaCl 0.9%, is poured in silicone glass tube. Using a syringe, connected with a 19 G Luer needle, pour below the level of the blood, one volume of 50% isotonic Percoll solution diluted in NaCl 0.9%. With the same identical procedure one volume of isotonic Percoll solution at 69% is poured below the 50% Percoll solution. Centrifuge tube at 350 x g for 20 min. The densities of the various isotonic Percoll solutions were determined using graduated density (r) beads (Pharmacia), and diluting each vial, from 1 to 9, with 1 ml of PBS. 20 µl of each contents were mixed together and placed at one volume of NaCl 0.9%. The specimen has been processed in the same identical way as the experimental of the sample. After centrifugation, the granulocytes band was removed with a Pasteur pipette and subjected at two washing with PBS, each followed by a centrifugation at 400 x g for 10 minutes. If erythrocytes contamination is present, eliminate it through osmotic lysis, resuspending pellet with one volume of 0.9% NaCl and add three volumes of distilled water, and delicately mixing for 30 seconds. At the end of time, add one volume of 3.5% NaCl in order to stop the reaction. After centrifugation at 400 x g the suspension is subjects to once wash with PBS, resuspended pellet in PBS and the recuperated cells are analyzed.

Neutrophils separation medium (NSM) one step

Polymorphonuclear leukocytes (PMNS) are prepared according to the procedure using NSM having density about 1.113 ± 0.001 g/ml, and Osmolality about 445 ± 15 mOsm (in two flash, dissolve exactly 13.8 g in sodium metrizoate in about 40 ml dH2O and exactly 8 g of Dextran T 500 in about 40 ml, and when full dissolved, mix both in continuous stirrer and fill up exactly to 100 ml with dH2O, filtered to sterilize using 0.22 nm filter, and stored at room temperature, protect from light). To use, 5 ml of whole blood drawn in centrifuge tube containing as anticoagulant 1.5 mg/ml of EDTA dipotassium salt and mixed gently, that than were carefully layered over 3.5 ml of NSM in a 15 ml centrifuge tube. The tubes were centrifuged (500 x g, for 30 minutes) in a swing out rotor, at room temperature. After centrifugation, 2 bands became visible, where the upper monolayer is mononuclear cells, and the bottom monolayer is polymorphonuclear cells. Erythrocytes and dead cells are found in the pellet. Using Pasteur pipette, the serum (top layer) and the upper cell band were carefully removed without disturbing the lower PMN band. Then the PMNs band are harvested and transferred to another 15 ml centrifuge tube and diluted with an equal volume of 0.45% NaCl. They are centrifuged (300 - 500 x g, for 10 minutes at 4 °C) and washed twice with sterile 0.1 M PBS at 4 °C. If the PMNs appeared contaminated with erythrocytes (pink color), 1 ml of cold sterile water was added to the cells for 30 seconds (to lyse the erythrocytes), then PBS was added for a total volume of 14 ml and centrifuged as above. The final cell pellet was resuspended in 1 ml of PBS and the cells are counted using a hemocytometer, and their viability evaluated using trypan blue exclusion criteria.

Image - Centrifugation of human blood and graduated density beads on an

discontinuous gradient of Percoll

I am trying to isolate neutrophils from whole human blood. I have few questions reagarding the reagents. Can you please tell me whether it is needed to maintain any specific pH for the 3% dextran in 0.9% NaCl? And how do you sterilize the dextran solution? Did you autoclave?

Thanks in adavance

2% Dextran T500: dissolve 10 g of dextran 500 in 500 ml of 0.9% NaCl and filter-using 0.22 µm filter to sterilize. Store the solution at 4 °C for up to 4 week, and pre-warm to room temperature prior to use. Not's necessary to maintain a specific pH of the solution. For to separate neutrophils from whole blood in citrate as anticoagulant, mix 1 volume of whole blood with 1 volume of 2% Dextran T500 solution. Gently mix by inverting the tube several times and allow to sediment at room temperature for 20 – 30 min. Erythrocytes, which are permeable to dextran, sediment more rapidly than leukocytes.

About in autoclave sterilization, dextran solutions may be also sterilized by heating in an autoclave. These solutions are stable for many years and are best stored at constant temperature. The optimal pH for storage is between 6 - 7. However, Dextran is stable at ambient temperatures for extended periods in the pH range 4 - 10. Other techniques for sterilization of Dextran solutions, in addition to filtration, is the irradiation process, may lead to degradation. During autoclaving a slight decrease in pH and a slight yellowing may be observed, which does not affect Dextran fraction solutions or performance. The autoclaving protocol for Dextran fraction solutions will depend on many variables, for example: • Size of container, • Container material, • Packing of the autoclave, • Duration of heating and cooling, • Other components present.

Thank you so much for the answer and the detail protocol. Thanks.

Durgesh, I've tried using your method and the RBCs lyse very well but when I stain my cells for CD16+CD14- I am only recovering about 40% cells; what purity do you think you're achieving and how do you confirm it? Many thanks

Dear Olivia,

Try HNP-1, CD66b and MPO to confirm presence of PMNs.

Yulianna Puspitasari was your issue of not seeing two bands solved ? I am also facing the same issue.