yield of PBMCs - (Oct/12/2007 )
Hi 
if any one can help me please ....i face realy i big problem....
after the isolation of the WBC (white ring ) from the ficolled blood or boffy coat
i freez the cells...at about 10 million cell per vial...and the method
of freezing is....after washing the cells and pelleting it out ,i lose the pellet
then i add 10% DMSO in FBS 1 ml / 10 M...then i store it in isopropanol box in -80C
then later i transfer it to LN.....the problem is that after thawing i get 3 or 4 Million cell
the yield is too low 
....(thawing prosedure...i put the vial in 37C water bath...till
small ice remains....then i put the cells in 9 ml of media serum free then i spent at 1500 rpm
for 8 min...)
Muna
if any one can help me please ....i face realy i big problem....
after the isolation of the WBC (white ring ) from the ficolled blood or boffy coat
i freez the cells...at about 10 million cell per vial...and the method
of freezing is....after washing the cells and pelleting it out ,i lose the pellet
then i add 10% DMSO in FBS 1 ml / 10 M...then i store it in isopropanol box in -80C
then later i transfer it to LN.....the problem is that after thawing i get 3 or 4 Million cell
the yield is too low
....(thawing prosedure...i put the vial in 37C water bath...till small ice remains....then i put the cells in 9 ml of media serum free then i spent at 1500 rpm
for 8 min...)
Muna
Dear Muna,
Your problem is that the speed is far too far for centrifugation. Also RPM means nothing, you must represent your speed in G. The speed you need to pellet cells after cryopreservation is 100g i.e roughly 700-800rpm depending upon the radius of your rotor.
The greater the speed the more damage you cause to your cells.
Hope this is useful
Rhombus.
Actually i am 
sceared to loose the T cells...which later i need for the MACS ....and as you know the T cell is
smaller in size... 