Flow cytometric analyses disclose intercellular communications in FasL‐stimulated T cells: Results and trouble shooting

Abstract

LIFE depends on the ability of cells to communicate with one another. Much of this crosstalk occurs at cell-cell contacts and is regulated by complex structural interface: neurological and immunological synapses that transmit cell-cell signals through the extracellular space, relying on mechanisms of ligand-receptor signalling across tight cell-cell junctions. In addition to these well-known examples, other cellular structures involved in cell-cell communication have been identified (1). A frequent result of cell-cell communication is collective population behavior that can potentially lead to complex phenotypes not observed in separate individual cells, for example, in development or tumor formation (2,3). Immune responses against pathogens or any foreign antigens require fine immune regulation, where cellular communications are mediated by either soluble or cell surface molecules. Generally, absorption, exosome production and uptake, internalization, and membrane nanotube formation are the probable mechanisms through which the membrane fragments and cytoplasmic content are transferred from one cell to another (4). The identification of new structures involved in cell-to-cell communication has led to the development of new analytical and imaging tools, which have allowed us to enhance our understanding of the ubiquitous phenomenon of cell-to-cell communication. Such tools include quantitative cell microscopy, now mainly represented by two distinct techniques: flow cytometry (FCM) and image cytometry (IC) (5). FCM allows analysis of cell suspensions, cell-by-cell quantification of optical signals, rapid analysis (several hundred cells per second), and cell sorting, whereas IC allows precise localization and quantification of optical signals emitted by each point of the observed field and image analysis (morphology and morphometry). FASL STIMULATION OF T CELLSThe Fas/CD95 surface receptor mediates rapid death of various cell types, including autoreactive T cells, which can trigger autoimmunity. In this study we investigate novel aspects of Fas signaling that define a ''social'' dimension to receptor-induced apoptosis. At least in some cell types, FasL can be stored in granules and then rapidly released at the cell surface in response to external stimuli. Alternatively, FasL can be cleaved from the membrane by matrix metalloproteases and act as a soluble cytokine (6). In its membrane-bound form, FasL acts as a ligand for Fas and can trigger Fas-induced death.In our experiments, CD41 T cells were purified from PBMC by negative selection using the MACS system. CD41 T cells and Jurkat cells were both treated with FasL at a final concentration of 0.5 lg/ml for a maximum of 2 h. Our previous findings (7) showed that Fas stimulation led to an intriguing (and previously unsuspected) asymmetry of death propagation according to cell conjugation and rapidly induced extensive membrane nanotube formation between neighboring T cells. The experiments were performed using flow cytometric (FC) and morphologic (fluorescence and ti...

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“…After centrifugation (13,000 rpm for 10 min at 4°C), pellets were resuspended in 0.5 N NaOH for the determination of proteins using Coomassie brilliant blue reagent from BioRad Laboratories (CA, USA) [18]. are presented as mean FI ratios of the HFEtreated samples and the controls from three separate experiments ± one standard error of the mean [19,20]. Comparisons between HFE-treated and untreated samples were evaluated using the Student t-test.…”

Fas Signalling Promotes Intercellular Communication in T Cells

“…As we have recently demonstrated [32] is important to evaluate also cell conjugates, because the formation of stable cells conjugated, i.e. detectable by flow cytometry, is a pre-requisite for the cell-to-cell communication and the subsequently exchange of cellular material.…”

“…Previously Poupout [30] , [31] have used stringent gating strategy to remove cell conjugates from the count of cells exhibiting exchange of specific dyes. As we have recently demonstrated [32] is important to evaluate also cell conjugates, because the formation of stable cells conjugated, i.e. detectable by flow cytometry, is a pre-requisite for the cell-to-cell communication and the subsequently exchange of cellular material.…”

“…This increased from 0.2 to 1.8 µg of protein for 10 7 cells. More recently, we applied flow cytometry to study the release of membrane vesicles from primary T cells [32] . The flow cytometry approach consists of mixing untreated and Fas treated CD4+ T cells with beads of defined size (Ø 1 µm, 2 µm, 5.2 µm) to obtain a size calibration of small particles (the majority falling within gate R1 in Fig.…”

“…The significant inhibition obtained with latrunculin and nocodazole suggest the importance of the actin and microtubules in the TNTs formation in our experimental model. We are mindful that flow cytometry cannot properly evaluate the presence of TNTs in primary T cells, which require accurate morphological analysis for proper detection [28] , [32] , [41] . For this reason, we have paired up the flow cytometry studies with some confocal microscopy data ( Fig.…”