Have you checked by blood smear or cytospin that your samples do contain neutrophils?  Sounds silly, but this would be my first check.  Do you have access to a differential blood counter?  If so, how many neutrophils do you expect?  When I separated horse blood for PMNs and monocytes, many years ago, I had only 2 percoll layers and put the anti-coagulated blood directly on top.  One collection was between the different percoll densities and the other from the top, below the serum.  The red cells were just spun to the bottom in one step. 

Perhaps too many layers are complicating things.  Perhaps just try 2 densities which will isolate neutrophils only, then add the monocyte separating layer to that, after it works.

Yeah, the samples do contain neutrophils, as I've managed to get some before. Unfortunately I don't have access to a differential blood counter but I have been making slides using the cytospin with my final cell populations once I've isolated them. Yeah I've tried the 2 layer technique as well, but that doesn't seem to separate properly. The protocol I'm using for the 2 layer says spin for 20 minutes at 400 x G - is it possible that I should be spinning it harder for longer?

Thanks for your help!

Longer might work, but I'd be careful about harder.  Have you changed centrifuges at all?  The equation for g is 11.17x (r) x (w/1000)squared  where r =radius and w=rpm.  might be worth checking.  For horse blood (not really of use to you I know....but anyway) I used 60% and 75% percoll centrifuged at 200g for 15 mins at 4oC.  Is the percoll kept in the fridge and is the centrifuge cooled?  This could alter the density, if not.  I've just found some density information.  60% percoll is 1.07g/ml and 75% percoll is 1.09g/ml (Lees, Dawson and Sedgwick. (1986) Equine Vet.J., 18(6), 493-497).  If you know the density of dog PMNs and MNs in iso osmotic fluid you should be able to work out the best percentages to use.

Strasser et al., Veterinary Immunology and Immunopathology. 62 1998 29–35.

Might be worth a try.

Abstract

A simple method for the simultaneous separation and purification of peripheral blood mononuclear cells PBMC and polymorphonuclear neutrophil cells PMNC was developed for comparative and functional studies in the immune system of the dog. Purity and cell viability were 95%, yields were similar to those obtained by other techniques but without red blood cell contamination. Differential blood cell count studies of the isolated cells in blood samples of beagle dogs and German shepherd dogs demonstrated that the 1.077/1.119 double density centrifugation is an effective method of acquiring both highly purified blood mononuclear cells and polymorphonuclear cells as separate entities from the same sample. The interface between plasma and 1.077 contained an average 97% blood mononuclear cells vs. 3% polymorphonuclear cells, and the interface between 1.077 and 1.119 an average 96% polymorphonuclear cells vs. 4% blood mononuclear cells. These data indicate that Histopaque 1.077/1.119 double density gradient allows the purification and physical separation of lymphocytes and phagocytes from a blood sample in the dog, enabling the investigator to examine both cell types from the same sample simultaneously.

Thanks Janet, I'll give that one a try. We haven't changed centrifuges but the temperature may certainly be a factor. All of my protocols said room temp but perhaps I'll also try it cooled to 4 C. Really appreciate the help guys, thanks!

Hi, I have a question.

Who do you prepare the different concentrations of Percoll (80%, 75%, 70%, 65%).

You dilute in RPMI?

Thanks

You should dilute in an iso-osmotic medium.  RPMI should be fine or 0.9% saline, PBS etc. Anything suitable for cells or cell culture. 

Hi Ruby Coates I realize this thread is a bit old, but any chance changing your protocol to 4oC help out with your isolation? I'm also having issues isolating immune cells using percoll densities and was wondering if setting up my gradients and spins at RT was maybe hindering my cell yields.