Counting Nuetrophils and monocytes using Trypan blue

[Bijou]

Hi,

Neutrophils and monocytes are my cells of interest, I am having trouble counting the cells... I am using a X2 diultion (10microliters cell suspension + 10microliters Trypan blue)... I can't clearly see the cells. Should I increase the volume of TB used? Is there an alternative method of counting?

We have an automated cell counter (CASY), however, cell diameter is an issue as manytimes after gradient separation I do not end up with a completely homogeneous population.. thus the CASY ends up giving me a higher count that the actual number of neutrophils I have. \

Thoughts?
Thanks,
Amina