I tried to isolate neutrophil from human venous blood followed the protocol as below:
1. mix 10ml blood with 10ml Hank's balance salt solution (HBSS) without Ca and Mg. Stand the tube for 30 min to let RBC residue.
2. Transfer the RBC-free supernatant onto 5ml Ficoll layer, centrifuge at 1350rpm/30 min.
3. Discard supernatant. Lyse the remained RBC in RBC lysis buffer (0.15M NH4Cl, 10mM Na2HCO3, 0.5M EDTA, pH 7.4), centrifuge for 10min. Repeat this step twice until no RBC remained.
4. Wash cell pellet twice with HBSS (without Ca and Mg)
6. Re-suspend cells in RPMI containing 0.2% Bovine serum albumin.
All the steps and buffers are kept at room temperature.
After isolation, i saw lots of cell dead (just only 20% cells survived). Moreover, when I seeded the cells on culture plate, they easily made clumps.
I need the cell lysate for Western Blot, but it was hard to extract the protein from the clumping cells...
Does anyone have any suggestions on how to prevent cell death and clumping?
Is there something missing from your description of "1."? Where is the sugar to agglutinate the erythrocytes? (eg dextran or HetaSep)
In any case, take a look at a sample of your cells at each stage to work out where the death is occurring. I would also recommend that you have everything at 4ºC/on ice from the ficol step.
In my experience the best protocol for isolating neutrophils in good condition is that described in J Immunol Methods. 1990 Jun 12;130(1):15-8. "A method for isolating neutrophils from moderate volumes of human blood." McFaul SJ.
In our hands cells isolated in this manner have good viability and can be stimulated, eg with FMP, to give good levels of response.
You need to be extra careful about the media you use to isolate neutrophils. Are all the media that you use cell culture grade that is free of endotoxin? We only use the top quality endotoxin-free (or at least endotoxin-tested) cell culture grade media for isolating neutrophils. Neutrophils are exquisitely sensitive to LPS and endotoxin, and will readily activate and clump in response to trace quantities of these bacterial products.
Also, we use dextran to sediment RBCs in the first step, then extract the relatively RBC-free plasma (supernatant) and put this on a Ficoll gradient to be spun at room temperature. This is another critical step - if you spin the cells at a cold temperature (4 C) through Ficoll, neutrophils will also clump and die. It's important to spin the neutrophils through Ficoll at room temperature.
Clumps may indicate you are centrifuging at too high a gravitational force.
We do an initial dextran sedimentation (Dextran, Cat #: BP1580-100, Fisher BioReagents)
Then RBC lysis
Then Ficoll 1077 centrifugation (Lymphocyte Separation Medium). Neutrophils are in the pellet, mononuclear cells in the interface.
We use D-PBS w/o Ca2+, Mg2+ (tissue culture grade) for all steps and for storing the neutrophils on ice after separation. RPMI plus BSA can activate neutrophils.
In this thread there were also many reasons for clumping discussed:
https://www.researchgate.net/post/Is_there_an_advantage_to_using_EDTA-coated_tubes_rather_than_heparin-coated_tubes_for_collecting_human_blood_to_purify_granulocytes?
Neutrophils (PMN) can become activated during the separation procedures. If so, they will generate oxidative products and degranulate, which will, in turn, damage other cells.
Endotoxin in your buffers can serve to activate neutrophils. I had that experience working in Mexico. Make sure you make your buffers with low-endotoxin water, or buy 1X buffers commercially. Over-vigorous RBC lysis can also damage and activate PMN. Finally, I agree with Stephan-keeping the cells cold during preparation will limit the chances of spontaneous activation, damage and death.
Clumps may indicate that there was too many, too long and/or too high RPM centrifugation. The used RCF shuld no exceed 1400 x g, but recomended 1000 x g. Max. cumulated centrifugal time shuld be under 50 minutes, 40 or 30 more recomendend.
An other important question: what is the rate of preparated cell vs. all aviliuable cell in the blood sample?
A third possible cause that the preparation itself takes too much time. In my experiments too long separation times (e.g. blood delivery from hospitals) limited some functional aspects of controll PMN.
And here is a separation protocol, which works in our lab:
Mix blood samples (35-40ml) with citrate and with 5ml 6% dextran, leave it for 30 min. at room temp.
Collect PMN enriched plasma, centrifugate it with 1300 x g for 10 minutes
Use Ficoll-paque to separate mononuclear cells from PMN (1000 x g for 20 minutes, with slow desceleration), PMN will be in the sediment
Lysate red blood cells with 5 ml distilled water, no longer than 25 secundum
Stop lysis with concentrated NaCl (end concentration sholud be 0,9%)
Pool down PMNs with 1000 x g for 10 minutes, and pick them up in the sollution, what You want to use for experiment (eg. HBSS, PBS, medium-H, etc.)
To Charles King: It's important not to keep the neutrophils cold during the Ficoll spin though, because they clump if they are spun at 4 C in Ficoll. Otherwise, yes, the neutrophils should be kept on ice throughout.
The Gold standard technique for the purification of neutrophils is dextran sedimentation and discontinuous plasma-Percol gradient separation (C. Haslett, L. A. Guthrie, M. M. Kopaniak, R. B. Johnston, Jr, and P. M. Henson. Modulation of multiple neutrophil functions by preparative methods or trace concentrations of bacterial lipopolysaccharide. Am J Pathol. Apr 1985; 119(1): 101–110. PMCID: PMC1888083). This method has also been used for the purification of murine neutrophils and is therefore applicable for small volume preps too (1. Juss JK, Hayhoe RP, Owen CE, Bruce I, Walmsley SR, Cowburn AS, et al. Functional redundancy of class I phosphoinositide 3-kinase (PI3K) isoforms in signaling growth factor-mediated human neutrophil survival. PLoS One. 2012 Jan;7(9):e45933). We have used these methods consistently for many years in our lab, to produce reasonable purity (>97%), minimally active neutrophils for functional studies, and have had no issues with high numbers of dead cells. A key aspect of purifying neutrophils for in vitro studies, is the avoidance of separating them from autologous plasma, which even for a short time significantly alters their functionality. It also means that any data derived from cell lysates will be artificially altered.
A word of caution wrt to BSA - a large number of available sources have significant LPS contamination, so you may want to review the one you have been using to ensure this is not the case.
Red blood cells can be lysed with distilled water instead of NH4Cl. The protocol is identical except that contaminating erythrocytes are lysed by a 20-s hypotonic shock in water (10 mL), and normal osmolarity is reconstituted by the addition of 10 ml HBSS (2X).
Thank you very much for all your replies.
May I ask other questions:
1. Would using gradient Percoll help to avoid RBC lysis step?
2. How to make hyposonic water for RBC lysis? Could I use only distilled water?
3. Can we exclude LPS from buffer by filtering through a 0.22um filter?
4. Can I use RPMI for maintain the cells for 24hr? Is there any other appropriate medium for neutrophil? Do I have to add serum to medium?
I hope to get suggestions from you again.
Thank you.
The plasma-Percoll gradient density centrifugation step is preceded by a dextran sedimentation (can also use 6% hydroxy-ethyl starch) which separates mixed white cells from red blood cells, removing the ned for a lysis step (both hypotonic and ammonium chloride lysis have been shown to activate neutrophils).
Approx 80% neutrophils will undergo spontaneous apoptosis by 20 hours when cultured ex vivo. Our way of culturing neutrophils for 24 hours is at 1x10^6/ml in Roswell Park Memorial Institute medium-1640 (RPMI), 10% heat inactivated fetal bovine serum with 20 mM HEPES, and penicillin and streptomycin in Flexiwell plates at 37degrees C in a 5% CO2 atmosphere.
Hope this helps.
Dear Le Duy Pham,
I have successfully isolated neutrophils from human blood. I had initially encountered with same problem. Please find the following protocol, you will definitely acquire more than 90% survival. (ALL THE NEXT 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 regard!
Durgesh
Dear LeDuy Pham,
the other contributors have said all relevant things (to my knowledge). But there is one thing left to be commented. Polymorphprep works pretty fine if you use blood from healthy volunteers. Moreover this method does is not such stimulating to the PMNL such the red blood cell lysing method. But if you want to analyze PMNL from for example severely injured patients or patients suffering from severe systemic Inflammation, you will notice that the PMNL and PBMC fraction cannot be distiguished clearly from each other. FACS-analysis revealed that a lot of contaminating PBMC can be found in the putative PMNL fraction and vice versa. In that case we apply a Ficoll Gradient, remove carefully everything above the red cell pellet and apply erythrocyte lysing with ammoniumchloride-EDTA-solution.
Best regards
Dirk
Dear LeDuy Pham,
neutrophils are most sensitive for any changes, they are sensitive for their separation, among others. Still, we suceeded to separate and plate them and even examine membrane fractions with Western blots after removing cetached cells.
You can download our paper from here and GoogleScholar:
Major laminin-binding and F-actin-linked glycoproteins of neutrophils.
M Keresztes, Z Lajtos
Cell Biology International 10/1997; 21(9):543-50.
When you need just to examine native neutrophils, the best is to do immunolabelling in wgole blood and then flow cytometry as quickly as you can after hemolysis (removeing red blood cells) . and then do virtual isolation of neutrophils by the software of the flow cytometer.
Most of my research has been related to neutrophils, so hopefully, I can help you if you need further advice.
All the best,
Margit Keresztes
Thank you Margit,
In your article, the method for isolating neutrophil is described briefly. I greatly appreciate if you could describe more detail. It would help me a lot.
Hope to receive your advice.
Thank you.
Thank you all,
Finally, I got it.
I found that the main problem was RBC lysis step. Follow my previous protocol, the cells are exposed to RBS lysis buffer for 10min, 2 times. With the new protocol, I replaced RBC lysis buffer by distilled water and stopped the reaction by adding HBSS 10X after 20sec incubate.
All steps were proceeded at 4oC and I avoid pipetting up and down.
Recovery >95% and very few cell clumps were observed.
Thank you for your all helpful suggestions.
Excellent, thanks for letting us know what the problem was. Just as an added note, there is more than one way to do water lysis on the cell pellet. We always use 1.5 ml sterile water to lyse the pellet for just a few seconds (10-30 sec), and gently pipette up and down with a 2 ml steripette, followed by transfer of the water to 12 ml RPMI containing EDTA (5 mM). The tube is then capped and gently inverted to make the solution isosmotic. Centrifugation brings cells down and these can be resuspended in appropriate serum-containing and supplemented RPMI.
Also, we usually rest our neutrophils for an hour before experiments as they seem to be quite refractory to stimulation immediately after they have been purified. So I recommend that the neutrophils are rested on ice for an hour before you proceed with experiments.
Thank you so much Paige Lacy,
Could you please confirm that after isolation, you do keep neutrophils suspended in RPMI on ice for 1hr, not at 37oC?
Thank you again.
Yes, they are suspended and kept on ice (sideways, not necessarily immersed completely) for 1 hour.
Thank you so much Paige Lacy. Hope to receive your kind suggestions in future.
We use the following protocol for isolation of neutrophils.
1. Add 1 ml of ACD (acid citrate dextrose) to a 15 ml centrifuge tube.
2. Add 5 ml of venous blood to ACD.
3. Then add 3 ml of 6 % Dextran made in 0.9 % NaCl solution. This is to enhance rouleaux formation. The dextran solution is made by adding 6 gm of Dextran powder to 100 ml of 0.9 % NaCl.
4. Mix well gently – 20 times.
5. Keep for 1 hour.
6. Take the supernatant and the interface into another centrifuge tube. Tilt the tube while taking the supernatant.
7. Centrifuge for 12 minutes at 1200 RPM at 4 degree Celsius.
8. Pour out the supernatant.
9. Add 3 ml of millipore water and mix with the pipette to lyse the RBCs. Then add 0.6 ml KCl within 25s to bring back the osmolarity to 300 mosm.
10. Then make up the volume to 12.5 ml by adding phosphate buffered saline (PBS) and mix well.
11. Centrifuge at 1300 RPM for 6 minutes at 4 degree celsius.
12. Redo the steps 8, 9, 10 and 11 two or three times to get rid of the RBCs.
13. Suspend the pellet in 0.7 ml of PBS.
14. Take 0.8 ml of Ficoll into a centrifuge tube and layer the cells suspended in PBS over it.
15. Centrifuge at 1500 RPM for 30 minutes at 4 degree celsius.
16. Aspirate or pour out the supernatant.
17. Suspend the pellet in 0.6 ml of HBSS.
Good luck.
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