I think that the water will affect PBMC also. As for the neutrophils there is a method of (I think) glicogen injection i.p. and after few days you can isolate a lot of neutrophis from peritoneum.

I have good success to remove eries from blood by the following protocol, but this is not specific for isolation of neutrophils

Material:

EDTA-tube (0,1 mol/l EDTA pro 10 ml Blut), Eis, Zentrifuge

Erythrozye-Lyses buffer:        1,55 M NH4Cl                     82,91 g/l

100 mM KHCO3                 10,01 g/l

10 mM EDTA                       2,92 g/l

PBS:                                       1X PBS Ca++/Mg++ free

NaCl:                                      0,9% (w/v) in H2Odd

Procedure:

1.     Collect 1 ml blood and put on ice.

2.     Add 30 ml of Erythrozye-Lyses buffer, mix carefully, and incubate for 10 min at room temperature.

3.     centrifuge (1000 xg = 3600 rpm, 10min, RT) and resuspend cell pellet in 20 ml  1X PBS Ca++/Mg++ free.

4.     centrifuge (1000 xg = 3600 rpm, 10min, RT) and resuspend cell pellet in 1 ml  physiological NaCl solution.

Dear all,

Thank you very much for all your suggestions.

I am sticking with BD FACS lysis buffer as it gives me approximately 10-18% neutrophils which I confirmed by using CD11b+Ly6-G + staining. 

@Carl Fortin- Yes indeed mice have very small population of neutrophils in peripherral blood. 

HI Jyoti Pant ! I have the same problem trying to lyse RBC from mouse blood with BD lysing buffer. I have a lot of erythrocytes still present in my samples when I go to the cytometer... Could you please give me some advice? Do the murine erythrocytes need more time to lyse? Maybe agitation helps? OR maybe higher Lysis buffer:blood proportion? Thank you!