Current Core Research
1. Signaling analysis using the ArrayScan VTI
Figure 2
The core has worked extensively on characterizing signaling events in primary human myeloid cells, including macrophages and dendritic cells, after stimulation with various PAMPs and DAMPs using the ArrayScan imaging cytometry system. The laboratory has demonstrated that the ArrayScan can accurately measure cytoplasm to nuclear translocation of NFB when compared with the standard EMSA method (5) (Figure 2).
In addition, the ArrayScan provides valuable morphometric parameter measures (nuclear area for example) on each cell analyzed. The ArrayScan offers a number of advantages over the EMSA method for nuclear translocation studies. The analysis is quantitative, is non-radioactive, is relatively inexpensive per analysis, can be done rapidly, requires very low cell numbers (5-10,000 cells per well), provides multiparametric, cell-by-cell measurement values including morphometry, and is completely automated. This type of analysis can be easily applied to dissect PAMP and DAMP activated signaling in a variety of adherent cell types, including primary myeloid cells. The VTI allows the use of up to 6 different fluorescent channels making multiplexed translocation assays feasible. For example, the laboratory has measured the simultaneous translocation of NFB and STAT-1 in response to IFN + IL-1 (Figure 2).
Figure 13
The typical protocol for analysis of a transcription factor would be to fix the cells at various time points with 2% PFA, permeabilize with Triton X-100, and stain with primary antibodies against the transcription factor, then stain with the appropriate secondary against either rabbit or mouse IgG. Each secondary is conjugated to a different fluorophore allowing for simultaneous analysis of up to 5 different molecules. Using the ArrayScan, the laboratory has shown that dendritic cells and macrophages have differential NFB nuclear translocation kinetics in response to stimulation with LPS (Figure 13).
The PMA-differentiated macrophage-like cell line THP-1 showed comparable response kinetics as primary M-CSF differentiated monocyte-derived macrophages. Both primary macrophages and PMA differentiated THP-1 display abundant NFB nuclear translocation with as little as 10pg/ml of LPS present. This serves as a robust and highly sensitive assay for the presence of LPS and can potentially be used to measure the activity of other PAMPs and DAMPs. The assay can be readily performed in a high-throughput screening arrangement using the THP-1 cell line.
Oxidative induction of Autophagy
(Blue, Hoechst stained nucleus
with Green LC3 Spots surrounding, 20X)
2. Quantifying Autophagy using Imaging Cytometry
The Core has developed an assay which can quantify the process of autophagy by counting the number of LC3 Spots in each cell. Experiments inducing autophagy by starvation, oxidative stress, and rapamycin have all shown an increase in autophagy as defined by the ArrayScans capacity to measure an increase in punctate LC3 staining. These results have been confirmed via Western.
