Analysis of cytokine induced phosphorylation of STAT3 in peripheral blood mononuclear cells by flow cytometric and western blot assays

Abstract

Signal transducer and activator of transcription (STAT) is a family of intracellular proteins that are responsible for carrying the signal from the cell surface to the nucleus in response to specific ligands. Once in the nucleus, STATs activate the transcription of specific genes. To date, seven human STATs have been identified. Among these STATs, STAT3 is considered as oncogenic. It activates genes that block apoptosis and inhibits antitumor immune responses (1). STAT3 is also essential in early embryogenesis and plays a role in cell growth and survival, differentiation and apoptosis depending on the target tissue. Analysing STAT3 signalling provides insights into pathology and can be used as a tool for diagnosis, prognosis and therapy development. Traditionally, western blot has been used to analyse cell signalling but it is impractical in analysing rare cell populations or providing information at the single cell level. Moreover, it is a demanding and time consuming technique that offers qualitative and less sensitive analysis. The rapid evolution in the multi-parametric flow cytometry and the availability of both epitope specific antibodies and sophisticated software facilitate the wide application of this technology in cell signalling studies. Flow cytometry has the ability to resolve different subcellular sets in a heterogeneous population, collects data at a single cell level and correlates multiple markers simultaneously. However, it requires highly standardized protocols for maximal sensitivity. The aim of this study was to assess the dose and the time response of both total STAT3 and pSTAT3 to in vitro stimulation with either IL-6 or IL-10 in peripheral blood mononuclear cells (PBMC). This assessment was done using both the flow cytometry and the western blot techniques. The results of this study showed that lower doses of IL-6 (1 & 10 ng/ml) were not sufficient to induce phosphorylation of STAT3. However, following stimulation with 100 ng/ml of IL-6, no significant change in the level of total STAT3 could be detected in either lymphocytes or monocytes from 3 different donors using either the FC500 or the Accuri cytometer. Using the FC500 cytometer, a small but insignificant increase in the pSTAT3 was seen in the lymphocytes and monocytes. A significant increase in STAT3 phosphorylation was only observed for monocytes after 15 minutes stimulation with 100 ng/ml of IL-6 using the Accuri flow cytometer. xii When the fluorescent labelled antibodies used in the flow cytometric assays were used for western blot probing, western blot analysis of stimulated cell lysates with 100 ng/ml IL-6 detects proteins of a low molecular weight than STAT3 or pSTAT3 which may explain the flow cytometric results of IL-6 stimulation. In IL-10 stimulation experiments, lower doses (1 and 10 ng/ml) tested by flow cytometric and western blot techniques demonstrated insignificant STAT3 phosphorylation induction. Following stimulation with either 50 or 100 ng/ml IL-10, no significant change in the total STAT3 was seen in either lymphocytes or monocytes when using the Accuri flow cytometer. However, stimulation with 100 ng/ml IL-10 induces STAT3 phosphorylation from 10 minutes through 30 minutes in both lymphocytes and monocytes. Longer times were required and high inter-individual variability was noticed for the activation of STAT3 after stimulation with 50 ng/ml IL-10. By using different antibodies from those used in the flow cytometric assay; the western blot results were comparable with the flow cytometric findings following stimulation with 100 ng/ml IL-10. The addition of phosphatase inhibitors during the flow cytometric protocol didn’t show any increase in the STAT3 phosphorylation. However, using paraformaldehyde for fixation and methanol for permeabilisation significantly decreased the mean fluorescence intensity of the PE conjugated antibodies comparing to the BD commercial fixation and permeabilisation buffers. The onset and the signal intensity of “in house” chemiluminescence mixture for western blot detection of STAT3 were comparable to the commercial ECL reagent used. However, the background of the “in house” mixture increased with time and was higher than with the commercial product. Upon longer exposure, the background increased enough to cause signal loss. In spite of the number of advantages of the flow cytometric assay compared to the western blot assay, these results are highly dependent on the specificity and the selectivity of the used antibodies. Furthermore, flow cytometry requires a highly standardized protocol to be able to assess the normal level of signalling proteins which could be later applied to detect abnormalities. It is suggested that the antibodies used in the flow cytometric assay be tested by western blot to confirm their selective detection of the target protein before their use in the flow cytometric analysis.