Thanks Lamaksha. I do have my doubts on the use of banked blood for neutrophil isolation, but was hoping it would be possible with freshly harvested banked blood. I am going to wait and see if anyone else can give me some pointers on this.

Thanks Peter.

Dear Dayanjan Wijesinghe,

It depends on the process for blood banking you use in your country (in general for transfusion).

By direct venous puncture, in general, transfusers want to isolate red blood cells (RBC) and platelets; then conserved them for further transfusion. For this purpose, they use special medium (APDC) , and red blood cells ie are conserved at 4°C for about 3 weeks.

But in general, they want to avoid, or remove leucoytes, because at the origin of immunization of recepients; and blood concentration technics enrich RBC and clear leucocytes.

You have some particular manners to isolate preferentially neutrophyls from other cells; and also classicaly monocytes are more easy to isolate, in general due to their capability to adhere.

For polymorphonuclear cells, you have special medium to build particular gradients, with differential centrifugation (sucrose gradient or commercial solutions available); but as for monocytes, isolation conducts in general to celll activation (and indeed for moncyte, adhesion is the first step of activation). And often, isolation of netrophyls conduct to degranulation.

A last way is cell sorting and concentration after by selective sorting with monoclonal antibodies directed against specific markers (CD) of each cell category. But heavy technic because of the material required.

I hope this pannel of descriptions could help you.

Best regards

Didier J

We never work on blood taken over 24 hours ago to work on neutrophils

Thanks guys. This is what I have been trying to find. Much appreciate your help. My expertise in lipidomics in case any of you have a question related to that particular topic, I will be glad to help! Thanks again.

When I did isolation of monocytes and neutrophils i mainly used whole-blood obtained from a blood bank I used gradient centrifugation. However, this method also worked for banked blood, but the neutrophils where always a bit activated already from start, and you could not get nearly as much cells as with the whole-blood freshly isolated. Briefly, you put 15 ml of polymorpfprep in a 50 mL falcon tube, then you layer 10 ml Lymphoprep ontop, and finally layer 25 mL of the whole-blood on top (total vol. 50 mL/tube). Subsequently, you do a gradient centrifugation: 340 xg at RT for 40 min i in a swing-out centrifuge and besides your pellet in the bottom of the tube, you will obtain monocytes in the upper layer, and the neutrophils in the lower layer. Do not forget to wash and do RBC lysis for optimal purification. Isolated neutrophils will enter spontaneous apoptosis within 24 h, so always isolate the neutrophils fresh for the day of experiment.

Good luck!

I agree with Sebastian and Christoph.

And as I said, simple adhesion activates monocytes and also neutrophils (even centrifugation trigger also degranulation; you have to be gentle :-). In this way, the more neutral plastic tubes are necessary not to activate cells (some specific are sale, but to verify in your conditions)

Only "long-term" cultures are possible with monocytes (not a good term because long-term is only 3 weeks with monocytes) ; but not with neutrophils for which it is only in vitro survival.

If you absolutly need to have less activation as possible, as said by Christoph, you have to perform your experiment the day of isolation, in a short time.

And the protocol described by Sebastian is one of the classical ones used by neutrophil specialists, with differential gradient; necessitating a good practice to obtain a maximun of cells.

Best regards

Didier

I guess banked blood would require approval by some body - here we have to apply through our local governance system even for experimental use of donated blood within the Transfusion Service, and we certainly cannot give any out to collaborators not from the Transfusion Service even if it is approved for our own use. However you will need fresh cells - neutrophils must be taken from freshly donated venous blood immediately (they have a half life of hours) - so banked blood would not be useful. Check also that your transfusion service does not leukodeplete blood donations (we do). We can get useful mononuclear cells from buffy coats produced as a byproduct of platelet preparation (also from platelet pheresis cone residue) - but again these should be fresh (we can use buffy coats up to 16h after donation for mononuclear cell preparation). For neutrophil preparation from fresh blood, one of our local professors (C Haslett) developed useful procedures for getting minimally-activated neutrophils during his personal research days, essentially sedimenting red cells with dextran then employing percoll gradients:

Haslett, C., Guthrie, L. A., Kopaniak, M. M., Johnston, R. B., Jr., and Henson, P. M. (1995) Am. J. Pathol. 119, 101–110

Ward, C., Chilvers, E. R., Lawson, M. F., Pryde, J. G., Fujihara, S., Farrow, S. N., Haslett, C., and Rossi, A. G. (1999) J. Biol. Chem. 274, 4309–4318

Your neutophils and monocytes have different densities and will form bands at different places in step or continuous gradient centrifugation - for example with Percoll (GE Healthcare) or OptiPrep (Axis-Shield) - (Google these for methods). In my experience Polymorphprep step gradient can only be used on very fresh blood and you will always get some red cell contamination - maybe not important? Worth looking at the Axis-Shield methods (on their website) - you can also manipulate cell densities by changing osmolality - and make the cells distance from each other by that and by volumes in steps or slope of density gradient - so that you get purer cells with fewer steps. If you wash by centrifugation you will activate both packed neutrophils and monocytes, so you have to work around that too.

i don't know what exactly you want to do with these celles,

but you can try to seperate them by the immunmagnetic technique.

it means that you can use the microbilles like CD14 microbille pour select the monocytes,

there are positive selection or negative selection.

if you don't need very pure cells; it will be enough with the continnous gradient centrifugation.

Any antibody based technique like immunomagnetic beads or FACS risks activating the cells due to interaction of antibody with cell surface markers (some of which may be activation receptors) or at least leaves the cell "contaminated" with the antibody and its conjugate or bead.

We find that pelleting any cells activates them to some extent. Chris Haslett made some of his scientific reputation by coming up with a method for getting non-activated neutrophils, then looking at various activation sequelae. Basically you sediment the red cells quickly using dextran or hyroxyethyl-starch, leaving leukocyte-rich plasma which you take off and layer on a gradient to separate the leukocytes according to density (check out references to his method). He used percoll - not sure whether continuous or discontinuous gradient - or I think you could use OptiPrep (we tinkered around with it recently looking for reticulocytes and red cell progenitors in mobilised blood samples). The use of a hyper-dense band at the bottom of the tube catches your densest cells and prevents them pelleting. You could always clean the cells up with MACS by negative selection by taking out what you don't want from any fraction (the residual "wanted" cells shouldn't bind antibody/beads if you choose the right antibodies for depletion). I would recommend using unconjugated antibodies (cheaper) in cocktails, then removing all bound (labelled) cells with an anti-mouse-IgG bead (such as Dynal). Difficult to avoid centrifugation of cells for washing, but use low speeds and resuspend by gentle aspiration, not vortexing. I used the dextran sedimentation / ficoll (pellet) method (not lysis) to get neutrophils for chemotaxis experiments years ago - they performed fine, but were probably activated (that wasn't relevant). You should be able to design a one step (multi-band) separation - just use bigger volumes in each band to get good distance between cell layers. I guess you will check purity by flow cytometry. Avoid lysis if you can - or at least check out its effect on what you are studying.

As another alternative to RBC lysis, you can consider using the HetaSep (where this step is included in some EasySep column-free cell isolation kits). The HetaSep works by causing the RBCs to aggregate and settle to the bottom while nucleated cells remain suspended in the supernatant.

Instead of pelleting the cells by spinning at high speed, the protocol involves centrifugation at around 100 x g.

(For more info, please refer to http://www.stemcell.com/~/media/Technical%20Resources/0/F/6/7/9/29622_PIS_3_0_0.ashx)

From the supernatant (which also contains the granulocytes), you can proceed with cell sep protocol.

Hope this helps,

Dunstan

Dunstan Lim recommends essentially what I recommended above - "Basically you sediment the red cells quickly using dextran or hyroxyethyl-starch, leaving leukocyte-rich plasma". This technique has been around for at least 40 years, and until the recent introduction of GMP-grade ficoll was one of the main ways of getting GMP-grade leukocyyes from blood samples.