Hi Rocco,
I'am using a different protocol, very easy.
Neutrophils were separated by Percoll method modified (Eggleton, 1998). Percoll gradient was made by slowly addition of 3 mL of Percoll solution 70% ( Percoll 100% 2.1mL + 645 uL Hank’s balanced salt solution (HBSS)1X + 255 uL HBSS 10X) above 3 mL of Percoll solution 60% ( Percoll 100% 1.8 mL + 990 uL HBSS 1X + 210 uL HBSS 10X) . Following, 3 mL of whole blood was added slowly above Percoll gradient and was centrifuged 500 x g for 35 min at 22 ºC, with acceleration/brake 2m/s2. After the centrifugation, a halo between Percoll solutions 70% and 60% containing the neutrophils was observed and collected. Then, the neutrophils were washed with HBSS by centrifugation at 400 x g for 10 min at 22 ºC.
Considering your second question, I did a lot of experiments involving neutrophils and lymphocytes survival. Neutrophils come apoptotic cells in 18 hous (culture medium RPMI) charaterized by flow cytometry. About lymphocytes they come apoptotic in 48 hours (culture medium DMEM).
And about your third question, you can do a RBC lysis with differents protocols (NH4Cl solution - look for the protocol) and proceed the same protocol.
Best regards
This protocol has worked very well for us. Neutrophils activate extremely easily, so treat your samples as gently as if they were "time bombs". Do not run them up and down through pipettes if at all possible. Resuspend them by tapping and swirling, keeping on ice intermittently. How long do they survive in media - we saw about a 20% loss after 6 hrs, and around 60% died overnight. Best used fresh. Collecting the blood in ACD and not other anticoagulants is critical I have heard. You can also skip step one part 5 - it makes it more difficult to resuspend the pellet for RBC lysis.
Materials:
6% Dextran in 0.15M NaCl (ice cold, sterile filtered, Amersham cat # 17-0320-01)
Ficoll-Hypaque (room temp, GE Healthcare, cat # 17-440-02)
PBS (ice cold)
SQH2O (ice cold)
0.6 M KCl (ice cold)
***IT IS CRITICAL TO TREAT THESE CELLS AS GENTLY AS POSSIBLE***
Step One: Enrich for neutrophils by dextran sedimentation
1. Collect fresh blood from donors in tubes containing preservative-free ACD.
2. Mix 30 mL blood with 15mL 6% Dextran/NaCl solution in a 50-ml centrifuge tube and incubate in upright position 60 min at RT. Two layers will form.
3. Aspirate the leukocyte-rich plasma (top layer) and transfer to a new 50-ml centrifuge tube.
4. Pellet cells from the plasma by centrifuging 12 min at 1,150 rpm without braking, 4°C. Aspirate and discard supernatant.
5. Resuspend cell pellet in a few mls of ice-cold PBS by gently tapping and swirling the tube. Top off with cold PBS to 50 mls total, then centrifuge at 1,350 rpm, 4 °C, low brake for 7min.
Step Two: Remove RBC
1. To remove residual RBC, subject cells to hypotonic lysis. First, remove all but a little PBS from the pelleted cells. Resuspend the cells in this residual PBS by gently tapping and swirling the tube.
2. Add 6 ml ice-cold SQH2O for 30 sec. Then restore isotonicity by adding 2ml ice-cold 0.6M KCl. Dilute it up to 50 ml with ice-cold PBS.
3. Centrifuge 7 min at 1,350 rpm, 4 °C, low brake for 7min.
4. Discard supernatant. If cell pellets do not appear relatively free of RBC’s, repeat steps 1-2 once or twice.
Step Three: Remove mononuclear cells by Ficoll-Hypaque centrifugation
1. Immediately resuspend cell pellet in 2ml ice-cold PBS.
2. Layer cells over 4mL Ficoll-Hypaque solution using a pipette.
3. Centrifuge 35 min at 1,500 rpm, RT, without braking.
4. Resuspend cell pellet (NOT the cells at the interface, the ones at the very bottom) in 2ml prewarmed media, count and seed 1 x10^6 cells/ ml. Per 20 mls blood you can get 2-8 x 10^7 neutrophils.
FACS Analysis
Neutrophils can be seen as CD11b+ Gr-1+ or CD11b+ Ly6G+ (preferred)
Exclude monocytes and macrophages using F4/80 and Ly6C
Thank you very much! I will try following your suggestions!!
Best,
Rocco
For human neutrophil FACS analysis I'd exclude monocytes with CD14 and CD64 (though immature neutrophils express weaker levels of CD64, the monos are much brighter). CD11b is very similar on human peripheral blood neutrophils and monocytes. CD15 or CD16 with CD24 might be a good selection. Nice thing about using CD16 and CD24 together is that you can gate out eosinophils if you like (CD24+/CD16-/ slightly brighter CD45 than the neutrophils).
Shouldn't be macrophages in peripheral blood to worry about.
Yes, I think that the use of CD16b together with CD15 is a good combination for neutrophil identification including also CD14 to discriminate monocyte contamination.
I would like ask another question about neutrophil purification. Is it possible to have a cell preparation that could include both neutrophils/granulocytes and PBMCs? For example removing only RBC without proceeding with ficoll?
We use a granulocyte exclusion kit (from whole blood) that is like a Rosetta system from STem Cell Technology. It works really well!
One method used in our laboratory involves PBMC separation on a ficoll gradient. You are left with RBC & granulocytes. Separate granulocytes by addition of equal amounts of 6% dextran and PBS and wait for the RBCs to rouleaux and gravitate to the bottom of the tube. The supernatant contains granulocytes and a few RBCs. We have found that granulocytes last 24 hours if they are kept at room temperature.
Yes, use a more dense media, such as Histopaque 1119. Dead cells and RBCs pellet to the bottom, while are the WBCs stay at the interface. Most density separation media are osmotically stressful towards cells, so limit their time in it. Alternatively, you can use Percoll, which is iso-osmotic.
If you're doing exacting biochemical studies, I recently learned that ammonium chloride lysis solutions cause neutrophils to acidify. An alternative is to lyse with distilled water (30seconds) then restore isotonicity.
Hi Rocco,
I'am using a different protocol, very easy.
Neutrophils were separated by Percoll method modified (Eggleton, 1998). Percoll gradient was made by slowly addition of 3 mL of Percoll solution 70% ( Percoll 100% 2.1mL + 645 uL Hank’s balanced salt solution (HBSS)1X + 255 uL HBSS 10X) above 3 mL of Percoll solution 60% ( Percoll 100% 1.8 mL + 990 uL HBSS 1X + 210 uL HBSS 10X) . Following, 3 mL of whole blood was added slowly above Percoll gradient and was centrifuged 500 x g for 35 min at 22 ºC, with acceleration/brake 2m/s2. After the centrifugation, a halo between Percoll solutions 70% and 60% containing the neutrophils was observed and collected. Then, the neutrophils were washed with HBSS by centrifugation at 400 x g for 10 min at 22 ºC.
Considering your second question, I did a lot of experiments involving neutrophils and lymphocytes survival. Neutrophils come apoptotic cells in 18 hous (culture medium RPMI) charaterized by flow cytometry. About lymphocytes they come apoptotic in 48 hours (culture medium DMEM).
And about your third question, you can do a RBC lysis with differents protocols (NH4Cl solution - look for the protocol) and proceed the same protocol.
Best regards
Unpleasant memories of step gradient isolation of lymphocytes for me! There is a density gradient product for polymorphonuclears by Axis-Shield: http://www.axis-shield-density-gradient-media.com/products.htm . I have never used it, but would be tempted first before moving to step gradients.
In addition to what Evan Chaswick mentioned about distilled water for lysis, I use a method based on Beckman Coulter's automated method. 0.12% formic acid for 8 seconds, then a sodium bicarbonate based buffer to restore isotonicity. The scatter properties of the neutrophils especially are different depending on the lysis method used.
Neutrophils only spend 8-10 hours in circulation in the blood, and then some of them marginalize to tissues, so don't expect a long life span.
The method you choose is really going to depend on your research question. You could also use immunomagnetic separation, negative would probably work better than positive. What are you trying to test?
I think Polymorphprep solution is a good product to separate human granulocytes from whole blood. Neutrophils are short lived cells but can survive until 12 to 24 h in a classical culture medium without growth factor.
Dear Rocco,
A simple way of granulocyte separation with the mononuclear cells and without having contact with any chemicals is layering the 2 ml whole blood on Ficoll 1077 after 1:1 dilution with cell wash medium. Leave it to gravity for 40 minutes at room temperature and then collect the upper 800 microliters. This way you can have the neutrophils as much near to their native state, and because you do not have any centrifugation, cells will be in good shape. 40 minutes have to be exact timing for good results.
In our experiments, neutrophils are earliest dying cells, we try to do all of our experiments and clinical tests in six hours after the collection of blood. They start to show apoptotic markers (annexin V) on cell surface after two hours.
Thank you for the suggestion!
2 ml of whole blood are not too low to have a significant number of neutrophils? And, what about buffy-coats that generally are of 50ml? Can you increase the ml of blood over ficoll?
Best,
Rocco
Normal range of neutrophils in peripheral blood is about 3-7.7 x 10^3 cells per microliter, normally about 54-69% of the white blood cells. So it will depend on what your definition of "significant number" is.
Dear Rocco,
You may increase the amount of blood and Ficoll as much as you want, it all depends on how much cells you need for your experiment. The 2 ml is enough for us to make neutrophil function tests for one patient if the neutrophil numbers are not too low.
Best wishes,
Dear Rocco
The lost of the cells by separation is about 50%.
Attention: The viability of the separated cells of the cancer patients or of the patients treated with x-ray is markedly impaired in comparison with that of healty persons.
Check the viability of the cultured cells daily using trypan-blue exclusion method. If it is necessary, change the culture medium.
best
eva
Percoll gradient or Histopaque method, both works well to isolate neutrophils. You can get 5-7x106 in 5ml blood. They get activated fast and undergo apoptosis from 18-24hrs cultured in RPMI. Donot use ice cold reagents in isolating neutrophils as they respond differently to treatments in functional assays. and to answer your buffy coat question, if you mean the histopaque or percoll that comes with neutrophils, you just have to resuspend it with PBS and mix properly so that the percoll or histopaque gets diluted and spin, you will be left with clean pellet. BW
We had done an apoptosis experiment about 14 years ago and found out that if you keep blood at 4oC; more than 80%percent of granulocytes will have annexin V positivity on their surface after two hours. So before keeping your blood routinely at fridge, I suggest you to optimize storage conditions at your hands/facility for granulocytes before moving to any other stage of experiments.
I've used polymorphoprep from axis-shield and multiple density percol layers to seperate granulocytes. Polymorphoprep works well with healthy human blood, and my impression was that it was less activating than percol based protocols, however it is more tricky if working with people who are anaemic or have significant left-shift of their neutrophils.
I am using polymorphoprep as well. Especially good if you want to have PBMC and granulocytes from the same donor because they form two separate rings.
Dear Rocco! I have a quetion for you? What is the best way to separate the Leukocyte(white blood cell) from the whole blood? I want all the leukocyte ,not only the granulocytes.
I used polymorphoprep several times and went I did flow analysis, the cells were very auto fluorescence (compare to my normal percoll gradient separation).My lab has been using the same protocol for over 20 years now and works great. It's basically a dextran sedimentation followed by a percoll gradient. Works great with big and small amount of blood. Would be happy to share some papers showing the protocol.
You can also lyse the RBCs after Ficoll sedimentation of granulocytes. Our lab routinely lyses RBCs after collecting the pellet from Ficoll centrifugation, and uses ddH2O for this step (for no more than about 30 seconds). After this, we resuspend cells in 12 ml RPMI containing 5 mM EDTA to reduce clumping.
Like Isabel, I've also used Percoll cetrifugation to isolate neutrophils and it works very well. For RBC lysis I have tried a number of solutions but the best and least disturbing for neutrophil isolation has been NaCl hypotonic solution.
All of the answers involved the isolation of neutrophils from whole blood. If the use of a non-human animal such as a rat is ok, then the best way to obtain a huge yield of neutrophils is to inject IP a noxious agent, do washes of the IP contents at intervals, pool the washes and add them to large petri dishes. The neutrophils will adhere to the bottom of the petri dish while the lymphocytes will be subject to removal with a wash.
In the good old days, we took whole blood mixed it with saline and let it sediment in 10 ml pipettes. The buffy coat would the be removed and put onto a pipette that contained glass wool and eluted with saline. Lymphocytes would come out in the eluate and the nejutrophils would adhere to the glass wool. The neutrophils can then be eluted with BSS containing a small amount of Tween 80. Viability is about 8 hrs. These cells are very active in phagocytosis of any particle including charcoal, etc. Here is a reference (yeah somewhat old) that may assist you: Broxmeyer HE, Van Zant G, Schultz EF, Koltun LA, LoBue J, Gordon AS. Glass wool separation of rat polymorphonuclear leukocytes according to adherence and maturity. J Reticuloendothel Soc. 1975 Aug;18(2):118-24.
I forgot to mention that low temperature (below 10C) will affect the adherence of neutrophils to the glass wool columns. Best done at 25C (room temp). There are other methods which I have not used since I left that area some 35 years ago. Here are the references:
Wright CD, Mülsch A, Busse R, Osswald H. Generation of nitric oxide by human
neutrophils. Biochem Biophys Res Commun. 1989 Apr 28;160(2):813-9. PubMed PMID:2541713.
2: Rainard P. Adherence, spreading, and locomotion of bovine polymorphs: effect
of proteins and metabolic inhibitors. Vet Immunol Immunopathol. 1988
Mar;18(2):129-37. PubMed PMID: 3388760.
3: Dutta SK, Bumgardner MK, Scott JC, Myrup AC. Separation and identification of
equine leukocyte populations and subpopulations. Am J Vet Res. 1981
Jun;42(6):1037-9. PubMed PMID: 6974519.
for isolation of all leucocytes, we follow Dextran sedimentation followed by RBC lysis and washing steps works fine and its simple. For Neutrophils we use both ficoll hypaque and dextran sedimentation. It also depends you want the cells to check their function or to look at the expression of some molecules. For cell function of course you need to be careful as these are relatively fragile cells.
Hi kate!
what is SQH2O? i looked it up and saw it meant nuclease free water but, how come it cuase the RBCs lysis?
can i prepeare theese water by myself or i have to buy them?
thank you
also,
can Paige Lacy (ddH2O) and Azadeh Arabzadeh (NaCl hypotinic solution) please tell me what is the exact protocol for the RBCs lysis?
also, Gulderen Yanikkaya Demirel (sendimentation without centrifugation) - if you take the upper 800 microlitters after sedimentation with histopaque, will you have the granulocytes, not only other WBCs, since the granulocytes sediment with the RBCs to the bottom of the tube? if true- what is your protocol for lysis of the RBCs?
thank you all.
There is no need for RBC lysis because RBCs are at the bottom, grans are at the top part of tube.. You can have some mononuclear cells which you can gate out during analysis
thank you.
i have a problem with what you said because if you sediment whole blood cells with histopaque 1077 the RBC will sediment with granulocytes.
it also say so in the PDF of all the different gradients that histopaque has to offer.
if you use 1119 as well you will get the gran in a separate ring between the RBCs and the ring of the MNCs.
RBCs always sediment with granulocytes after centrifugation through Histopaque. We find that water lysis is very straightforward and has minimal effects on neutrophil viability and function. We have tried NH4Cl on ice for 15 min, but this usually renders granulocytes refractory to stimulation. I am not sure about the use of NaCl hypotonic solution as we have never tried this.
Hi Catisani,
After Blood withdrawal, the vacutainer should be maintained at room temperature (24-28). This will reduce RBC adhearance. The best way to isolate granulocytes for experiment is using sigma histopaqe1119. During this procedure its very critical that you maintaing the histopaqe temperature at 24-28 C (not 37C). If you desire to extract granulocytes and agranulocytes, you can overlay histopaque 1119 above histopaqe1107. This allows you to separate granulocyte from agranulocyte.
Critical point-
1. Use vacutainer instead of syringe for blood withdrawal. Using syringe you see some RBC lysis, which may activate your neutrophil.
2. Dilute blood with equal volume of Calcium free HBSS (you can add 0.25mM EGTA) before separation.
3. centrifuge at 24-28C for 40 min at 900 rpm.
After separation, wash with HBSS (24-28C, Ca free)).
Critical step - While pipetting you have to be very gentle as harsh pipetting would activate neutrophil.
you can use NH4Cl lysis at this stage.
Transfer the layer of granulocyte (neutrophil and eosinophils) to cell culture plates coated with heat inactivated FBS.
Incubate for 45 min (24-28C)
Critical point - usually use 5-6 ml of HBSS during this incubation.
Slowly drain out eosinophils into another centrifuge.
To check the quality you can use dextran beads or lysed RBS in the isolated eosinophils (they dont bind to these).
Now transfer the petriplate to ice and use ice cold (4-6C) to drain out neutrophils.
further handling should be done at cold contition untill you start experiment.
Use RPMI 10 % (heat inactivated FBS or heat inactivated autologous serum) for culture. you can check for activation by simply putting latex beads.
Ideally if your neutrophils are not activated, they do not engulp beads (you can check with flowcytometry or microscopy.
You can activate using PMS (5ng//ml) or LPS (1ug/ml).
You can culture non activated neutrophils till 48 hrs.
good luck for your experiments.
8 ml heparinized whole blood in a 10 ml injector will be remained in steril incubator with 5% CO2 two hours vertically. İnjector needle will be bended 90 degree. Neutrophils and monocytes containing sera on top of the injector will be discharged through the needle on top of the Ficoll. After centrifugation pellet will be contained pure neutrophils.
This method my favorite neutrohil extraction.
Hello,
I tried different protocols on whole blood and the more convienient for my experiments was the Lympholyte-poly (CEDARLINE) is endotoxin free and is quick.
Hello,
Lympholyte-poly is a ready to use mixture of sodium diatrizoate and Dextran 500. I chose it because it was quick, sterile and endotoxin free as I had to study PMN activation by flow cytometry.
With fresh heparinized whole blood kept at room temperature :
- carefully layer 7ml of whole blood on the top of an equal volume of Lympholyte-poly in a 15ml tube. You must avoid mixing blood with the Lympholyte-poly.
- centrifuge at 500g for 40min at 20°C
After centrifugation : you will have a pellet with the RBC and two leukocyte bands : the mononuclear cells are in the upper band at the interface between sample/medium, the PMN are in the lower band between the RBC and the mononuclear band.
Whatever protocol you use and no matter how gently you treat the neutrophils, their activation can lead to apoptosis. Consider adding (pyrogen free) superoxide dismutase to promote survival of activated cells.
Dear all, when you isolate neutrophils from whole blood how many PMN/ml do you obtain?
Please note that different brands of density gradient solutions have different apoptotic effects on granulocytes. There is need for a good validation protocol before you use any brand..
Hi Rocco,
I would recommed method devised by Ferrante and Bignold (J Immunol Methods. 1987 Jan 26;96(1):29-33. I spent 4 years using this method every week without a problem. Also check Bignold LP, Rogers SD, Harkin DG. Eur J Cell Biol. 1990 Oct;53(1):27-34 and Harkin DG, Gadd SJ, Bignold LP. Biol Cell. 1993;79(3):251-7 for any suggestions.
best wishes,
Damien
Hello Rocco,
I tried different protocols on whole blood and I would recommed this method: Isolation of Human Neutrophils From Venous Blood (Nauseff, 2007) From: Methods in Molecular Biology, vol. 138: Neutrophil Methods and Protocols Edited by: M. T. Quinn, F. R. DeLeo, and G. M. Bokoch © Humana Press Inc., Totowa, NJ.
Best regards
Hi Rocco,
neutrophils become apoptotic within 18 hours in culture medium
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