In-cell Western assay: a new approach to visualize tissue factor in human monocytes.
BACKGROUND
Tissue factor (TF) is an integral membrane protein essential for the initiation of the extrinsic pathway of hemostasis. A precise understanding of the TF regulation is still limited and dependent on the availability of methodological tools. Here, we describe a new approach for assessing TF amounts in human mononuclear cells (MNCs) by using the whole blood experimental conditions.


OBJECTIVE
In order to study TF antigen levels in human MNCs, we applied a quantitative immunostaining technique-- in-cell Western (ICW) assay using an Odyssey Infrared Imaging System.


RESULTS
The ICW assay of TF in resting or lipopolysaccharide (LPS)-stimulated human MNCs was performed. Several sample preparation conditions were tested, namely the plating of MNCs prior to immunostaining, paraformaldehyde fixation, and an adequate cell number was used in the assay. By the use of recombinant human TF standards, it was possible, for the first time, to measure TF amounts in LPS-stimulated MNCs as 0.09 +/- 0.02 ng and 0.43 +/- 0.15 ng 10(-6) cells of surface and total TF, respectively. The concentrations of TF in resting MNCs, however, were below the detection limit.


CONCLUSIONS
A novel TF ICW assay is a reproducible, time- and cost-saving method, which could become useful for studies in the fields of physiology and pathophysiology of human hemostasis.
Egorina
E M
EM
Department of Biochemistry, Institute of Medical Biology, University of Tromsø, Tromsø, Norway. egorina@fagmed.uit.no
Sovershaev
M A
MA
Østerud
B
B
D015153:Blotting, Western / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D008070:Lipopolysaccharides; D009000:Monocytes / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D015203:Reproducibility of Results; D013925:Thromboplastin / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D016707:Tissue Fixation; D014162:Transfection
D008070:Lipopolysaccharides; D034741:RNA, Small Interfering; D013925:Thromboplastin