Dear Durgesh,

First it depends from which sample you isolate your PMN. By contrast with blood monocyres, neutrophils are more fragile, and can just survive in vitro during 3 days maximum; they dont' divide in vitro.

Isolation activates them and they destroy themselves in this case because of the lot of enzymes inside cytosol, and progammed cell death triggered.

So, you can use commercial prep (a lot of available) and follow centrifugation advices of manufacturer, don't "stress" the cell. You didn't precise the cell type you need (human, animals …), but if you use you human ones , you can use a 5-10 ml blood sampling and can obtain about 30 million cells.

During isolation, always gentle centrifugations, gentle pipetting … and in medium without Ca2+ and Mg2+ (ie HBSS), these ions activating PMN.

After isolation, PMN can be put in classical culture medium for survival (RPMI1640, AB, L-glutamine, autolog serum…).

Hope usefull.

Best regards

Didier

Good.

I think you wouldn't be able to maintain them more. Except if a colleague has another method.

Regards

Didier

Dear Durgesh,

The in vivo circulating half-life of human neutrophils is approx' 7 hours in most of the literature. These cells are extremely fragile, activate after exposure to almost any physicochemical stimuli, and even trace amounts of agents such as LPS. 70-80% of human neutrophils cultured for 20 hours are apoptotic, leaving only a small sub-set of what are probably to be phenotypically different cells still alive at this time point.

The majority of researchers study the pathogen killing and phagocytic abilities of freshly isolated neutrophils, rather than cultured cells. We have done this in our laboratory for a number of years very successfully. For almost all experiments, there is no need to maintain neutrophils in culture, with the exception of apoptosis/cell death studies.

Thank you very much Charlotte.

G-CSF and GM-CSF are both survival factors. Adding anywhere from 1-50 ng/ml of either would keep them pretty happy for a day or 2.

Dear Durgesh, how do you maintain neutrophil live for 3 days successfully?  May you share the protocol with me, please?

Dear Robert,

Survival of neutrophils in the artificial condition (in vitro) is depends upon your method of isolation. Most of cells dies during isolation if not isolated properly.

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 practised in my Lab.

Try with this method.

Hi, is this the protocol you are using for isolation only? Do you add something after this which helps to maintain the cells for 3 days? I'm really looking forward to being able to keep my cells live for at least 12-14 hours. I'm currently  isolating my cells by using EasySep Neutrophil  kit. Do you have any suggestions on how I can keep my cells live after that?

Hi Riva,

After completing isolation of neutrophils from blood, I culture them in RPMI 1640 with 10% FBS. No any special protocol for culturing human neutrophils. Make sure that pH of media should be in a range of 7.3-7.4. Do minimum pipetting during neutrophil isolation, this will help you to maintain PMNs live at least 12-14 hr.

Best luck.

 Thank you, Durgesh.

any idea on half life of bone marrow-derived neutrophils???? thanks a ton in advance.

 Dear Prakash,

I do not have any idea about BM derived PMNs. Sorry.

no problem..thanks

Hello Durgesh,

I have never maintained  neutrophils for over than 24 hr since after 24 hr I observed lots of apoptotic cells (>50%). How about your case of maintaining for 3 and half days? Did you see increased apoptosis?

Another tip suggested by Prof.Paige Lacy is to let the cell rests on ice for #30 min after isolation and prior to transfer to 37oC. It helps recover the cells after stressful isolation process. Moreover, in the isolation buffers (PBS or HBSS), you can add 20nM of EDTA, which could further prevent the cells from activation and clumping. 

I'd like to know any experience from you also.

Best luck,

Duy.

Hi Duy,

Apoptotic status of PMNs also alters from donor to donor. What I have observed a perfect healthy donor (Who have taken proper sleep for two consecutive day before donation shows relatively more non apoptotic PMNs population (for at least 12hr). Cells starts showing annexin positive reaction from 5-7hr after isolation but both annexin and PI (i.e. apoptotic cells) starts from 12hr onwards. Suggestions from other colleague is true. I haven't introduced EDTA treatment in my culture but one can try this also.

Neutrophil isolation procedure also affects your PMN apoptotic status. More harsh treatment may results in a callus of dead PMNs at the end of isolation protocol. So take as much care as possible during isolation of PMNs.

I hope this may help you.

Good luck!

Durgesh 

Hello Durgesh,

I work with rat and isolate peripheral PMN with Histopaque-1077 and dextran solution (6%):

Place 3 ml of Histopaque-1077 in a separation tube (Greiner) and then add the same volume of whole rat on the top of the Histopaque-1077, making a two-step gradient (Histopaque-1077 and blood). Then, centrifuge tube at 400g, 4ºC, for 45 min. At the end of centrifugation, three distinct phases were found. Discard the upper two phases (plasma, mononuclear cell and Histopaque-1077), and add 2 ml of 6% dextran solution (MW 250–500 kDa) in PBS (without Ca and Mg) to the lower one, which is rich in neutrophils and erythrocytes. Next, vortex the suspension and incubate for 20 min in a water bath at 37ºC. Collect supernatant and centrifuge at 270g, 4ºC, for 10 min. Thereafter, lysis the red blood cells, centrifuge (400g, 4ºC, for 3 min) and resuspend cell pellet in 1 ml RPMI.

My questions are:

As I mentioned above, to isolate PMN, I incubate the peripheral blood with dextran at 37ºC. To avoid PMN activation, do you recommend me to incubate PMN with dextran at RT rather than 37ºC?

I have RPMI (cat # 31800-022) and DMEM/F12 (Invitrogen, cat # 10565018). Which one do you recommend that I use?

I need to isolate extracellular vesicles (exosomes and microvesicles) from PMN spent media. Therefore, I need to culture PMNs in media without FBS. My experiment plan is to culture PMN for 3 and 6 h and then activate them with LPS (2.5 ng/ml). Do you think that I need to have FBS in medium for 3 and 6 h culture?

Many thanks,

Reza

Hi Preena,

Would you share the animal species on which you are interested in studying vaccine response? Would you please share the article which you are talking about. Then I will be able to comment on your problem.

Regards!

Hi..

Ficoll gradient separation separation and then cutlure with 20ng G-CSF maintain the cells pretty happy for 2 days easily!

Best of luck!

Hi Preena,

If I am not mistaken, in the given study (PDF), researchers are preparing vaccines for cattle mastitis. They have generated anti sera against FnBPs in mice and checked for opsonizing property of sera.

Since they want to check effect of mice anti serum on cattle's neutrophils hence they performed experiment on bovine neutrophils. But if your study is related to canine purpose you can check opsonizing effect of sera on canine neutrophils.

Best luck.

Is there any literature on neutrophil isolation that i can read through?

Hi Marquis,

Please find following protocol. I have done some modification in it. You can find it in previous discussion of this question.

Article Neutrophil Isolation Protocol

Hola. Soy Romina de Argentina y trabajo en el área de al inmunología y onocología. Específicamente trabajo con neutrófilos polimorfonucleares de ratones BALB/c portadores de un tumor.

Necesito estimular la vía NOX-independiente con Calcio, para obtener NETs, peor no logro disolver el calcio en el RPMI. Alguien lo hizo?

Gracias

Durgesh Pitale I am currently only getting about 14 million cells per 20ml whole blood whereas some people are getting up to 60 million e.g. @Didier JAMBOU. The protocol I'm using is ficoll, 3% dextran for 1h, then rbc lysis using 10x PBS as you described. Why do you think that is? Thanks

Dear Durgesh,

May I ask have you ever done the neutrophil migration assay? Are you culturing neutrophils from human blood in 1640+10%FBS not Human serum?

Best wishes,

Zihan

Dear Zihan,

For neutrophil migration assay I followed, "Article A real-time assay for neutrophil Chemotaxis

Best wishes!

Durgesh