There are many points for trouble shooting:

- Buffy coats were mixed with an anticoagulant (preferably EDTA) or at least diluted in 1:1 ratio with PBS at RT;

- Temperature was kept at RT (~22°C)? (critical point for erythrocytes separation);

- 500xg for 35min at lowest acceleration and no brakes with the right (swing-out) rotor?

Yes. We layered the buffy coat on Lympholyte-poly (1:1) and centrifuged it at 500 xg for 30-35 min, RT and brakes-off.

Red blood cells should pass through the gradient, while neutrophils and monocytes should not enter into it and should separate in two bands. Unfortunately, all the components of buffy coat remained over the gradient.

Thanks for your answer.

How old was the blood and how was it kept?  Fresh samples kept at room temperature do best.  Holding the samples at 4°C, particularly overnight, can really change the density of the cells.

You can purify neutrophils from monocytes leveraging the adhesion to the plastic surfaces of the latter. You must dilute the buffy coat in warm PBS+fetal calf serum in a Petri' dish in 37 degrees thermostat for two hours. Retrieves later the supernatant that should only contain neutrophils.  

Hi Lucia, in case your question is still open. You have 2 ways. The less expansive one is to use two density gradients Leuko-Spin and LymphoSpin (https://pluriselect.com/density-gradient-centrifugation.html). First put the Leuko-Spin in our Reaction tube, on that you put carefully a layer of Lympho-Spin and on that your blood sample. Spin for 1.000xg for 30 minutes. Now you will see two rings. The upper one contains the PBMC and platelet. The lower one contains your granulocytes. Take as much as possible from the upper layer away and collect the lower phase.  Purity is about 95%

Second way, positive cell isolation: use our CD15 M-pluriBeads directly in your buffy coat to isolate your neutrophils.  More expansive but higher purity >98%

I hope that will be helpful

Michael