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Summary
Neutrophils are among the first cells to arrive on the site of inflammatory immune response, and their functions and mechanisms have been studied extensively in vitro. We demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media.
Cite this Article
Copy Citation | Download CitationsOh, H., Siano, B., Diamond, S. Neutrophil Isolation Protocol. J. Vis. Exp. (17), e745, doi:10.3791/745 (2008).
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Abstract
Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory immune response. Due to their key role in inflammation, neutrophil functions such as locomotion, cytokine production, phagocytosis, and tumor cell combat are extensively studied. To characterize the specific functions of neutrophils, a clean, fast, and reliable method of separating them from other blood cells is desirable for in vitro studies, especially since neutrophils are short-lived and should be used within 2-4 hours of collection. Here, we demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media that is a mixture of sodium metrizoate and Dextran 500. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended in buffer to desired concentration. When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability.Protocol
Neutrophil Isolation Protocol
- Bring all reagents to room temperature.
- Collect 5.0 ml of neutrophil isolation media in centrifuge tube. Carefully layer 5.0 ml of blood over the separation media. Perform this step slowly and carefully, and with the pipette tip close to the surface of the media to avoid mixing the blood and the media.
- Centrifuge at 500 RCF for 35 min at 20-25°C. The blood should separate out into 6 distinct bands: plasma, monocytes, isolation media, neutrophils, more isolation media, and the red blood cell pellet (Figure 1). If these bands are not clear, the separation process was not clean and will need to be repeated.
- Carefully remove the top three layers (plasma, monocytes, and isolation media) using a pipette. Dispose of these layers.
- Carefully pipette the layer of neutrophils and all of the isolation media beneath the neutrophils. Place the solution into a clean centrifuge tube.
- Dilute the neutrophil solution to 10 ml with HBSS without Ca2+/Mg2+. Invert the tube a few times to suspend the cells.
- Centrifuge the neutrophil solution at 350 RCF for 10 minutes. A red pellet should be present at the bottom of the tube, containing neutrophils and residual red blood cells (RBCs). Remove the supernatant with a pipette carefully so that the pellet is not disturbed.
- To lyse the residual RBCs, add 2 ml Red Cell Lysis Buffer to the tube. To resuspend the pellet, vortex the vial at a setting of 3-4. Avoid increasing the vortex setting above 4, since this may cause the neutrophils to activate. It may be necessary to vortex for several seconds, or to "pulse" the vortex to dissolve the pellet.
- Centrifuge the tube at 250 RCF for 5 min. Remove the supernatant with pipette. Repeat the lysing process if required.
- Add 500 μl HBSS without Ca2+/Mg2+ to each tube. Again, vortex to resuspend the pellet at a setting of 3-4. Dilute to 10 ml with HBSS without Ca2+/Mg2+.
- Centrifuge the tubes at 250 RCF for 5 min. Aspirate the supernatant and discard.
- Resuspend the pellet in 250 μl HBSS/HSA Solution (2% HSA). Cells may then be counted and adjusted to desired concentration.
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Discussion
The density gradient separation method is used to isolate human neutrophils from whole blood using a mixture of sodium metrizoate and Dextran 500. This method is based on the mononuclear leukocyte separation method by Boyum (1968) which was modified for neutrophil separation by Ferrante and Thong (1980).
After collection from a donor, whole blood may be anticoagulated with EDTA, citrate, or heparin. Since they are short-lived, neutrophils should be used within 2-4 hours of collection. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended to desired concentration.
If the six bands are not distinct after the first centrifugation step, the separation process was not clean and will need to be repeated. For clean separation, make sure that the isolation media has not expired or contaminated. Separation may also be unsuccessful if the blood donor has consumed alcohol or medications in the 72 hours prior to blood collection.
To prevent neutrophil activation during the separation procedure, it is best to use HBSS without Ca2+/Mg2+, since the ions have been shown to prime cells. Resuspension of pellets should also be performed slowly, and vortex settings should be kept in the low- to mid-range so that cells do not become activated.
When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability. Purity and viability can be assessed by labeling the cells with neutrophil-specific marker CD66b and trypan blue dye exclusion.
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Acknowledgements
Funding from NIH R01 HL56621.
Materials
| Name | Type | Company | Catalog Number | Comments |
| Lymphocyte Poly(R) | Reagent | Cedarlane Labs | PolymorphprepTM can be used as an alternative | |
| Hank’s Balanced Salt Solution without Calcium Chloride | Reagent | Invitrogen | ||
| Human Serum Albumin | Reagent | ZLB Bioplasma | ||
| Red Cell Lysis Buffer | Reagent | Roche Group | ||
| Centrifuge | Tool | |||
| Vortexer | Tool | |||
| Pasteur Pipettes and bulb | Tool | |||
| PolymorphprepTM | Reagent | Axis-Shield | Alternative reagent to Lymphocyte-Poly (R) | |
| Syringe Filter | Other | EMD Millipore | SLGV033RS | Millex-GV, 0.22 μm, PVDF, 33 mm, gamma-sterilizable |
| Hank’s Balance Salt Solution with Calcium Chloride | Reagent | Invitrogen |
References
- Boyum, A. Separation of leucocytes from blood and bone marrow. Scand. J. Clin. Invest. 21, Suppl 97. 77-83 (1968).
- England, J. M., Rowan, R. M. The assignment of values to fresh blood used for calibrating automated blood cell counters. Clin. Lab. Haemat. 10, 203-212 (1988).
- Garcia, A. Comparison of two leukocyte extraction methods for cytomegalovirus antigenemia assay. J. Clin. Microbiol. 34, 182-184 (1996).
- Ferrante, A., Thong, Y. H. Optimal conditions for simultaneous purification of mononuclear and polymorphonuclear leucocytes from human peripheral blood by the Ficoll-Hypaque method. J. Immunol. Methods. 36, 109-109 (1980).
Comments
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what is the rational for using whole blood instead of purified white blood cells or enriched neutrophils for NBT test???
Why must HSA and not BSA be used? Do you have references for the effect of ETOH and drugs on separation success, and mechanism of action?
BSA can activate neutrophils. Brian Siano, on behalf of Hana Oh and Scott Diamond
How many HBSS and how many HSA need for HBSS/HSA solution preparation?
hi, why do you use HSA? or what is the action of HSA in buffer?
so many things contradict in the movie vs your text
From JoVE editor: video and text are complementary parts of each video-article. They are not necessarily 100% the same. There are things that are better described in video than in text, and vice versa. I think in this case the video part is very comprehensive and provides a very clear demonstration on how to do the experiment.
Are there any other reasons why one will not get 6 bands of separation using Polymorphprep? My separation is still not perfect. Repeatign causes the monocyte layer to lower towards the neutrophils. Any suggestions? Donor blood is not affected as mentioned in your video.
Any suggestions of modify this protocol so that it will work with NHP blood?
Why is ²% HSA added in the last step? What is the advantage of doing that? And what happens is I dont add it?
DŒs HBBS for HBBS/HSA preparation contain Ca AND Mg or only Ca?
I believe the question above refers to the last step of resuspension of neutrophils. At this point you could use various types of buffer solutions that suite your specific experimental goals. If trying to create a suspension of neutrophils in a physiological saline buffer, both Ca and Mg can be added.
I notice that the HBSS contains no phenol red in your neutrophil isolation method. I have noticed that phenol red is avoided (both during isolation and during experiments) in a number of papers implementing neutrophil isolation techniques. Any idea what phenol red dŒs to neutrophils? I have used a variation on the isolation technique (generously shared by Drs. Oh, Siano and Diamond) and ended by suspending my isolated neutrophils in RPMI with phenol red. Although phenol red might not be the (only) culprit, I am not seeing any neutrophil killing activity (ADCC assay).
I plan to attempt the method provided in this video later this week. Any ideas about phenol red?
We avoid pH indicator dyes because they often cause autofluorescence and lead to high background in fluorescent assays. We don't have any direct measurements of fluorescent dyes on neutrophil function.
I have seen that during neutrophil isolation from my blood there is no layer of neutrophils while there is a thick band of ruptured RBC at that position,every time.Can anyone suggest me,what could be the reason for that?
I'm getting the same result every time. Did you ever find out what's causing a thick band of RBC at the position of neutrophils bands?
I have been in face of the same problem that part of RBC can not sediment to the bottom band. This almost happpens when centrifuging at 500RCF but hardly happen at 450 RCF. So I guess 500 RCF is inappropriate.
Is this protocol applicable to Dog and Rat Neutrophil isolation? If so, do I still use HSA?
I have a similar question. I am interested in using this protocol to isolate neutrophils from mouse whole blood. Did you ever find out if this protocol works for rat neutrophil isolation?
Hi. It is said in the article that the neutrophil yield would be 2x10^6 per ml. Does that mean 2x10^6 per ml of whole blood used? So using 5 ml of blood (as used in the article) would yield 10 million neutrophils?
I got an extremely low count for my neutrophils. about 2 million from 5ml blood. Can you please help me troubleshoot?
Also, why did you mention 10-15ml blood (yielding x10^8 cells) in the beginning? Is it that you carry out the isolation from 10-15 ml blood in 2 or 3 parts of 5 ml each? Please help.
Thanks.
I a following this protocol, but running into clumping of the cells. Is there any way to prevent this from happening?