Adipose Digestion

[Scooter Hutchison]

I am trying to isolate macrophages from adipose tissue.  I have read numerous methods for this.  I am using Krebs Ringer Buffer (but no BSA).  After mincing the perigonadal fat (~ 3 grams) thoroughly, I am digesting the fat in ~ 10mg of type I collagenase (Worthington) in ~ 8.5 mL of Krebs for 40 min on an orbital shaker at 250Hz at 37 degrees.  I then use some chiffon mesh as a filter and spin down the non fat layer at 2000 rpm for 10 min.  After this is done, there is a VERY small ring of what appears to be RBC's.  But there is not a discernable pellet.  Can anyone tell me where I am going wrong?  Or perhaps give some ideas as where this procedure can go wrong?  This is my dissertation... so i NEED SOME HELP!  Thanks
 
Alexander