Cytometric analysis of protein expression and apoptosis in human primary cells with a novel microfluidic chip-based system.
BACKGROUND
Work with primary cells is inherently limited by source availability and life span in culture. Flow cytometry offers extensive analytical opportunities but generally requires high cell numbers for an experiment.


METHODS
We have developed assays on a microfluidic system, which allow flow cytometric analysis of apoptosis and protein expression with a minimum number of fluorescently stained primary cells. In this setup, the cells are moved by pressure-driven flow inside a network of microfluidic channels and are analyzed individually by two-channel fluorescence detection. For some assays the staining reactions can be performed on-chip and the analysis is done without further washing steps.


RESULTS
We have successfully applied the assays to evaluate (a) activation of E-selectin (CD62E) expression by interleukin-1beta in human umbilical vein endothelial cells (HUVECs), (b) induction of CD3 by phorbol-12-myristate-13-acetate in freshly prepared human peripheral blood lymphocytes, and (c) staurosporine-induced apoptosis in HUVEC and normal human dermal fibroblasts.


CONCLUSIONS
Results obtained with the microfluidic system are in good correlation with data obtained using a standard flow cytometer, but demonstrate new dimensions in low reagent and cell consumption.
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D017252:CD3 Complex / Q000096:biosynthesis; D002478:Cells, Cultured; D019040:E-Selectin / Q000096:biosynthesis; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism; D008832:Microchemistry / Q000295:instrumentation* / Q000379:methods / Q000592:standards; D014176:Protein Biosynthesis; D012984:Software; D013194:Staining and Labeling / Q000379:methods; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D017252:CD3 Complex; D019040:E-Selectin; D007375:Interleukin-1; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate