The protocol I am using is also with dextran and ficoll and I get a highly enriched population of neutrophils. The only difference, which I think it is why it doesn't work for you, is that I am using fresh blood. Once one centrifuge blood for an enriched buffy coat, one loose most of the neutrophils that go down to the erythrocyte layer.

you could try this. It worked for me.

Dear Agersew,

you could try this protocol by Sigma-Aldrich.

Hi, Lilli,

I think your protocol makes the cells' membranes too permeable and cells themselves leaky. Several suggestions based on our use of DCF-DA:

1) dissolve the dye in DMSO (do not dilute it)

2) vigorously stir or, if adherent cells, shake, while adding a small volume (no more than 10 microliters), calculated so its final concentration in cell medium is 1 micromolar and DMSO concentration is below 1% (that's enough to permit entry into cells without damaging them)

3) shorten the incubation to 30 min.

4) detach the adherent cells by scraping gently with a rubber or plastic tool without using trypsin (we needed intact cell surface proteins so never used trypsin to detach) though a brief exposure to 10uM EDTA in isotonic saline, followed by washing with saline, also works.

Good luck

Hi, you can try to use the Monopoly Resolving Medium( ICN Pharmaceuticals, USA), is a  density gradient of a neutrophil isolation medium. The protocols is similar to the ficoll but the separation is better.

Marina Conrado

Dear Agersew, 

I have been isolating neutrophils from mouse buffy coat layer regularly using Histopaque 1077/1119 gradient. Briefly, centrifuge whole blood @ 500g, RT, 15 min. Collect the buffy coat layer and dilute 4 times with RPMI. Prepare density gradient using 3 ml of Histopaues (follow manufacturer instructions). Layer the diluted buffy coat over the gradient. Centrifuge at 700g for 30 min with no brakes mode in swing out rotors at RT. You will see a faint white band at the interface of the Histopaque gradient. Collect it carefully with a Pasteur pippete ( Dont worry if few RBCs tag along). Dilute this layer in 4 ml RPMI. Centrifuge at 350 g for 5 min. Remove supernatant and add 2ml RBC lysis buffer (Sigma). Incubate for 30 s. Add 4 ml PBS and centrifuge again at 350g for 5 min. The RBCs will be lysed and pure neutrophil population will be pelleted. Collect and observe microscopically. Hope this helps!