Hi everyone,
I did some staining using Pharmingen anti-Bcl2-PE kit, with matched
isotype control (hamster Ig). The cells were mouse lymph node dendritic
cells.
Each sample was split and one half used for specific Ab, the other for
isotype. The Bcl-2 signal in this experiment is not bimodal but there is
single peak that shifts depending on exp. conditions. Therefore I
defined Bcl-2 protein levels as geo MFI (anti-Bcl2) minus geo MFI
(isotype) for each sample separately (each sample compared to it's own
background).
Although the comparison in Bcl-2 levels between different experimental
conditions is qualitatively OK (perfect correlation with apoptosis
markers), I am stuck with the following:
Some cells downregulate Bcl-2 to extremely low levels, i.e. even below
the MFI of the matched isotype. The calculated relative MFI is therefore
a negative value. Although the overall methodology is as strict as I can
conceive, I'm sure a negative expression level would seem odd for
reviewers. How should I deal with such issue?
Thanks for any help,
Karim
PS:
I looked in the archive and found an interesting discussion concerning
isotype control (summary posted by Barbara Breithaupt). Mario's answer
was especially enlightening. With this staining kit, I think I stuck as
much as possible to the criteria he mentioned concerning optimal use of
isotypes ("...this means that we would have to use a control antibody
that is (1) the exact same isotype; (2) conjugated to exactly the same
degree; (3) has the same background binding characteristics as your
antibody; and (4) is used at the same concentration. Rarely is more than
the first criterion met.").
PPS:
The protocol I used was:
1. FcR block
2. surface Ag staining, wash (PBS/BSA/EDTA/azide)
3. Cytofix/Cytoperm
4. anti-Blc2 or isotype (used according to manufacturer: undiluted,
quantity adjusted as function of cell mumber) + saponin, Permwash
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