Karim, If you add a small amount of a 3% Saponin solution at the same time you add your antibody from Pharmingen, this should eliminate your concern about loss of permeabilization. I normally add about 10µL per million cells in a total volume (before antibody) of 100-200µL. Randy Fischer TherImmune Research Corporation 9700 Great Seneca Hwy Rockville, MD 20850 (240) 453-6256 RFischer@therimmune.com > ---------- > From: Karim Vermaelen > Reply To: Karim.Vermaelen@rug.ac.be > Sent: Friday, August 11, 2000 2:54 AM > To: Cytometry Mailing List > Subject: bcl-2-PE staining kit: no saponin in ready-to-use > solution > > > Hi everybody, > Small technical question: > I'm using the Pharmingen bcl-2-PE staining kit for the detection of > murine bcl-2. Cells have to be fixed and permeabilized of course. > Next, > the infosheet suggests "20 µl of MAb solution per million cells". > However, the buffer in which the ready-made MAb is diluted contains no > permeabilizing agent. Could this have an impact on the penetration of > the antibody? (I got very poor signal). How fast does the cell > membrane > "close" again after witdrawal of perm-buffer? Is it safe to override > the > method on the infosheet and dilute the MAb in a saponin-buffer? How > far > should these ready-made MAb solutions be diluted? It'd be great to > hear > from anybody with experience in such staining protocols. > > >
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