Hi all,
I’m trying to purify monocytes from mouse spleens. However, in these preps an unusually high percentage of monocytes are dying, and thus I’m not getting a good yield for the purification.
My protocol follows:
Resect mouse spleens
Force mouse spleens through a 100 micron filter with a syringe plunger (I’ve also tried enzymatic digestion, using a syringe to pipet up and down to break up the spleens, and using scissors to dice the spleens)
Wash spleens in cold PBS
ACK lysis for 2 minutes
Wash 2x in cold PBS + 1 mM EDTA
Use stemcell mouse monocyte isolation kit (19861)
Count monocytes (at this point they’re all dead, based on trypan blue coloration)
Essentially, all my monocytes are dying during this procedure.
Can anyone point out where my protocol is going wrong? Does anyone have any suggestions?
Thanks,
Michael