Imaging analysis of STAT1 and NF-kappaB translocation in dendritic cells at the single cell level.
Rapid assessment of immune or stem cells, which are now widely applied in the clinical setting of cancer treatment, is necessary to speed their development and to determine their quality. We have evaluated immature dendritic cells (iDC) by semiautomated imaging cytometry which provides detailed assessment at a single cell level. Nuclear translocation of NF-kappaB was studied by imaging analysis as well as electrophoretic mobility shift assay with an excellent correlation (r=0.981) over a broad range of lipopolysaccharide (LPS) concentrations. Imaging analysis was time saving (5 h vs. 3 days), and required 30- to 100-fold less cells per analysis. Single cell information revealed remarkable heterogeneity between individual iDC and permitted detection of responses to 40 pg/ml of LPS. In IL-1beta/IFNgamma activated iDC, STAT1 responses preceded NF-kappaB responses, and the expression of both was strongly correlated in individual cells (p<0.001). IFNgamma amplified IL-1-induced NF-kappaB responses. NF-kappaB responses to IL-1beta, CD40L, and LPS were donor-dependent (n=7), correlated with the quality of iDC preparations (p=0.002), and IL-12 p70 production (p=0.010). NF-kappaB measurements in iDC within mixed cell cultures (iDC, NK, K562) demonstrated that these strategies are applicable for analyses of complex cell-cell interactions. Imaging analysis is a method that could be valuable for quality control of cell therapy preparations.
Vakkila
Jukka
J
Molecular Medicine Institute, University of Pittsburgh, 300 Technology Drive, Pittsburgh, PA 15219, USA.
DeMarco
Richard A
RA
Lotze
Michael T
MT
D000970:Antineoplastic Agents / Q000494:pharmacology; D023201:CD40 Ligand / Q000378:metabolism; D002450:Cell Communication / Q000502:physiology; D002467:Cell Nucleus / Q000502:physiology*; D002469:Cell Separation; D064987:Cell- and Tissue-Based Therapy; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003713:Dendritic Cells / Q000166:cytology / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D018664:Interleukin-12 / Q000096:biosynthesis; D020014:K562 Cells; D007694:Killer Cells, Natural / Q000166:cytology / Q000502:physiology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D016328:NF-kappa B / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D011786:Quality Control; D050794:STAT1 Transcription Factor; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D015534:Trans-Activators / Q000378:metabolism*
D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D007375:Interleukin-1; D008070:Lipopolysaccharides; D016328:NF-kappa B; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D015534:Trans-Activators; D023201:CD40 Ligand; D018664:Interleukin-12; D007371:Interferon-gamma