1. Bcl-2 monoclonals. The most widely used monoclonal antibodies for detecting human Bcl-2 protein are MablOO and Mabl24, both mouse IgGx, which are available from DAKO, Inc. (20). The 4D7 monoclonal (21), available from PharMingen, Inc., is also a mouse IgGj and is suitable for FACS analysis. The 6C8 antibody is a hamster IgG2a (22) (PharMingen, Inc.). For FACS-based detection of mouse Bcl-2 protein, we have used the 3F11 monoclonal with success (23,24), a hamster IgG2a (PharMingen, Inc.).

2. Control antibodies. As controls, the same amount of mouse IgGj (in the case of DAKO antibody or 4D7 antibody) or hamster IgG2a (in the case of 6C8) are added to duplicate cell samples.

• mouse IgGj negative control (Dako, Inc., cat. no. 931)

• hamster IgG isotype standard (Pharmingen, Inc., cat. no. 11091D)

3. Primary antibody optimization. For optimal results, the amount of primary antibody employed should be titered for each type of cell undergoing FACS analysis. Using too much can reduce the signal/noise ratio. For example, based on titration experiments performed using leukaemic B cells from patients with chronic lymphocytic leukaemia (B-CLL), we have determined that 0.32-0.65 |xg per sample of anti-Bcl-2 antibody MablOO (DAKO, Inc.) is optimal for FACS quantification of Bcl-2 protein levels, whereas 0.32 (xg was optimal for the anti-Bcl-2 antibody 4D7 (PharMingen, Inc.). Note that these concentrations are 5-10 times less than those recommended by the manufacturers.

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