A recent review  states that the development of phospho-specific intracellular detection necessitates development of reagents and adapted protocols. Nuclear translocation of transcription factors can be usually measured by immunoblotting of isolated nuclei by electrophoretic mobility shift assay (EMSA), or more recently in flow cytometry on purified cells [4, 27]. Aubry and coworkers described in 2002 an original technique to study the NF-κB pathway by multichannel flow cytometry after permeabilisation of DCs stimulated with LPS . Next, our group contributed in 2003 an original approach to study the phosphorylated transcription factor NF-κB in purified B-cells populations originating from blood or lymphoid tissues, with the need to purify the target cells to homogeneity. The main problem of this technique is the loss of information because of not studying cellular events under physiological conditions. Now, we understand the influence of the culture environment on any cell and the importance of studying cells within their context. So scientists need more and more techniques which study specific cells in their environment. In this report, we have developed and validated a flow cytometry to study transcription factors for various cell subpopulations within one cell type or a tissue suspension (we chose PBMCs), because there is a need to study rapidly, commonly and reproducibly such parameters (nuclear factor phosphorylation/translocation) in blood samples representing steady state or pathological conditions. Phosphorylation of proteins in signal transduction is traditionally studied by means of Western Blot Analysis and/or ELISA, which carry valuable information regarding homogeneous or purified cell populations. The present study proposes an original method to study phosphorylation of proteins in complex, heterogeneous, cell populations such as unseparated PBMCs. This method could be further validated to be applied to whole blood samples obtained from patients presenting with inflammatory diseases, for example, where there is a need to study cellular events . Indeed, the NF-κB pathway plays a pivotal role in immune and inflammatory responses and has attracted much interest as a novel target for the treatment of inflammatory states. Meanwhile, the major interest resides in the identification of cell subsets displaying altered nuclear factor phosphorylation/translocation, thus to be clearly identified by surface membrane characteristics.
Multiparameter flow cytometry offers the valuable advantage of characterising, almost without doubt, certain cell surface markers to identify subpopulations in response of a stimulus. In this study, we used 3 labelling conditions: one for each cell type, one for DNA gating and one for the intracellular factor of interest. In this manner, we marked only one cellular type and one intracellular factor per tube. However, by using a flow cytometer with more excitation lasers, it could be possible to mark DNA, all types of cells and the 2 intracellular factors in the same tube. Here we confirm that intracellular pathways of NF-κB and STAT3 can be rather easily monitored by Western Blot and ELISA (we thus took advantage of these reproducible techniques to set up optimal conditions for the stimulation of PBMCs with external signals leading to the use of either NF-κB or STAT3, that are for instance IL1β and sCD40L (NF-κB) and IL10 (STAT3), according to a general consensus . The development of a multiparameter flow cytometry technique permits us to decipher which of the stimulated cell population studied responded to treatment. We evidenced that IL1β and sCD40L should be used at 50 ng/mL for 30 min and IL10 at 100 ng/mL for 30 min. From the above experiments – principally aiming at presenting a convenient technique for studying and foremost tracing cellular translocation of – for the time being NF-κB and STAT (but – hopefully – additional pathways such as other STATs and also SMAD) – we could provide evidence that unseparated monocytes/macrophages responded strongly to sCD40L and B-cells to IL10. This result is not surprising because the literature showed the activation of PBMCs with these cytokines . This study has no real impact on the results but on the technique, itself. The obtained results proved this method is valuable to study activation of cells.
In flow cytometry, only 4 hours are necessary to prepare the samples and roughly 1 hour to acquire and analyse the data. It is the most feasible technique, however, the use of the cytometer and analyse software require training. Flow cytometry is not the most sensitive technique but the sensitivity is largely acceptable (1 μg/tube). Relating to the reproducibility, we observe a difference of the basal rate of activation depending on the individual samples. Regulated degradation of specific proteins is part of the intracellular biochemical changes that contribute to the regulation of signal transduction pathways; NF-κB activation thus, requires the rapid degradation of IκBα, the inhibitor of NF-κB. Before activation, NF-κB is sequestered in the cytoplasm by forming a complex with IκBα, the nuclear translocation signal of NF-κB being masked by the inhibitor. NF-κB can be activated by a number of stimuli, which trigger the phosphorylation of IκBα and lead to the rapid dissociation of NF-κB from IκBα. Once released from the IκBα complex, NF-κB immediately translocates from the cytoplasm to the nucleus, where it mediates the transcriptional activation of genes. At this point of phosphoprotein translocation, it is difficult to see the effective translocation by flow cytometry. 7-Amino-Actinomycin D (7-AAD) is a convenient, ready-to-use nucleic acid dye that can be used in flow cytometric assays for exclusion of nonviable cells, or to gate nuclei and label nucleic acids, in replacement of propidium iodide (PI) in flow cytometric assays . The advantage of 7-AAD over PI is its ability to be used in conjunction with phycoerythrin (PE) – and fluorescein isothiocyanate (FITC)-labelled monoclonal antibodies in 2-color analysis, with minimal spectral overlap. Therefore, the relationship of basal degradation to induced degradation remains uncertain . Moreover, NF-κB regulation of IκBα transcription represents a delayed negative feedback loop that drives oscillations in NF-κB translocation. Transcription of target genes depended on oscillation persistence, involving cycles of RelA phosphorylation and dephosphorylation . The oscillations in NF-κB signalling can explain the background levels of pNF-κB/pSTAT3 positive cells in unstimulated PBMCs. Nevertheless, a similar difference between stimulated and non stimulated cells is always detected.
This paper describes a novel flow cytometry method which allows the detection and quantification of intranuclear levels of the transcription factors NF-κB and STAT3 in distinct PBMCs subpopulations; this technique enables the identification of CD3, CD14 and/or CD19 subsets – within unseparated PBMCs (and, maybe in the future, whole blood cells) – which actually phosphorylate intranuclear proteins upon appropriate stimulation. These strategies will enable the development of simple though robust blood methods to identify normal signalling pathways as well as impairments in phosphorylating proteins in association with various human disease states (including acute and chronic inflammatory conditions), or with other chronic diseases characterized by an alteration of the cytokine homeostatic milieu, as seen in profound allergies or in cancer states .
In this way, our data stress the differences that are detectable between lymphocytes and monocytes regarding NF-κB or STAT3 phosphorylation upon relevant stimulation; an explanation could be because monocytes are associated with innate immune responses faster than, for example, T or B-cells, an issue which would deserve closer examination.
In summary, to determine the optimal conditions of activation, ELISA is the most practical technique to study at the same time a large quantity of samples. Once the optimal conditions have been identified, the Western Blot and the flow cytometry technique become the most adequate techniques, although the flow cytometry remains the most rapid method.