It could be neutrophils getting confused and blowing themselves up, along with your CD34s.  Do you see any evidence of viscosity in your samples, or foci of cell attachment to the glass? 

The neutrophils are unpredictable.  Tiniest changes in temperature, chemicals, vibration, even noise can push them over the edge, and make them freak out and blow everything up.  If that's what happens, be super gentle, super clean, and keep them cool.

Hi,

Thanks for your help! Rodolphe, the process has been kept the same for a long time...

Thanks John! When I thaw the MNCs, there is always some viscosity and even some clumps that I remove before going to the column. After the column, the sample appears fine, with no strange viscosity...I am usually careful with the MACS buffer to keep it cool while working.  Still, I will keep this in mind and be as careful as I can.

Hi, Marcia

I had same problem and used a little different protocol from yours.

Here is my thawing medium and protocol;

Thawing medium; DMEM-LG, 1%PEN/STREPT, 0,1%DNase (10mg in 10mL DMEM-LG for DN25 sigma product)

Protocol; (all procedure on ice) frozen cells dissolved immediately in 37C water, then transfer in 50 mL tube, add 100 uL thawing medium and gently shake for 3 minutes, then add 200uL thawing medium and gently shake for 3 minutes, then add 400uL thawing medium and gently shake for 3 minutes, 800uL..., 1600uL, Repeat the protocol until the medium 20 ml. then you can centrifuge the cell. So that aggregate formation is prevented

Also my MACS Buffer;

%0.5 BSA

2mM EDTa

PBS

I hope it helped. Good luck with,,