Expression of Tissue Factor on monocytes

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Hi Everybody

I am having som difficulties trying to set up an assay for measuring the
expression of Tissue Factor on monocytes labelling with a directconjugated
a-TF. I get a lot of background with my gammacontrol (unstimulated MFI 36,
stimulated MFI 60) (unspesific staining?) and just a little more specific
staining than my background (unstimulated MFI 32, stimulated MFI 90).

I stimulate whole blood with whole bacteria (Eqv. 2,8 microgram endotoxin
pr. ml blood) for 2 hours at 37 degrees. If I stimulate for 4 hours I loose
a lot of cells.

My questions are:
-Is the small increase in specific staining compared with background as much
as I can get with that stimuli?
- What can I do to reduce background (tried blocking with immunoglobulins
without noteworthy improvement)

Thanks in advance for any advice!

Regards
Dorte Christiansen
Researchlab
Nordland Central Hospital
Bodoe
Norway

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