Afterwards, they were incubated with 5 L of each antibody for 15 min at 4 C in the dark
Afterwards, they were incubated with 5 L of each antibody for 15 min at 4 C in the dark. Plasmon Resonance Imaging (SPRi), an optical technique allowing label-free and real-time analysis commonly used to detect specific interactions between different molecules [23,24,25,26]. In doing so, Milgram [26] described an antibody microarray using SPRi detection to simultaneously monitor in real-time T cell secretions of both IFN- and IL-2 after cellular mitogenic stimulation. The biochip was composed of microarrayed antibodies specific to T lymphocytes (anti-CD3e, anti-CD28), antibodies specific to secreted cytokines (anti-IFN-, Angiotensin 1/2 (1-6) anti-IL-2), and with a combination of both antibodies. Bindings of cells Angiotensin 1/2 (1-6) and/or cytokines with grafted probes Angiotensin 1/2 (1-6) were detected by SPRi. Although these systems provide promising advances, the cell secretions of cytokines are detected over a short incubation period, typically after 1C6 h of cell incubation around the biochip [18,19,20,21,25,26]. Since the kinetics of cytokine synthesis and secretion differ among cytokines [12,13,25,27], the analysis of cytokines on a short incubation time limits the ability of these techniques to perform functional analysis of cells according to cytokine secretions. This applies in particular to studies aiming to provide a complete Angiotensin 1/2 (1-6) characterization of functional modulations which can arise from certain cell cycle phases, varying half-life of secreted cytokines, and the variations regulation of cytokine productions. In the present work, based on the aforementioned biochip analysis format previously described [26,28], we have developed a system capable of handling the monitoring of cytokine secretions upon continued long-term cell culture of viable T lymphocytes. To achieve this goal, a strategy based on T Rabbit Polyclonal to Keratin 18 cell-specific and cytokine-specific antibodies was used: T cells injected around the biochip via a fluidic system were first captured around the microarray surface through specific antibodies recognizing their differentiation markers and afterwards, the cytokines secreted by captured T cells were detected by conversation with their specific antibodies grafted in close vicinity of secreting cells. Cytokine secretions were recorded either by fluorescence labeling with a specific antibody after 24 h of on-chip cell culture or by direct measurement of SPRi signals for up to 65 h. 2. Experimental Section 2.1. Materials and Reagents Phosphate buffer saline (PBS), phorbol 12-myristate 13-acetate (PMA), phytohemagglutinin (PHA), ionomycin, trypan blue, absolute ethanol, sodium dihydrogen phosphate (NaH2PO4), sodium phosphate dibasic (Na2HPO4), sodium hydroxide (NaOH), sydrochloric acid answer (1N HCl), paraformaldehyde A (PFA), and glass slides coated with transparent and electrically-conducting film of indium-tin-oxide (ITO), were purchased from SigmaCAldrich (St. Quentin-Fallavier, France); whereas N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES), AIM-V Serum Free Medium, and Dulbeccos 1X PBS without calcium and magnesium were purchased from GIBCO? (Cergy Pontoise, France). Human ELISA IFN- and Human ELISA IL-2 Ready-Set-Go (10 96 Assessments) protocol kits were purchased from eBiosciences (Rennes, France), LIVE/DEAD? Cell Vitality Assay Kit and dimethyl sulfoxide (DMSO) from Molecular Probes (Cailloux-sur-Fontaines, France?), Vivaspin filter membranes from Vivascience (Palaiseau, France), Angiotensin 1/2 (1-6) Thiol-PEG: HS-C6-(CH2-CH2-O)6-OH; from Prochimia (Sopot, Poland), PDMS and its curing brokers Sylgard 184 from Dow Corning (Seneffe, Belgium), and thermal paste (Silicone Heat Release Transfer Compound Thermal PasteWhite) from DX DealExtreme (Paris, France). Human monoclonal antibodies used for the capture of T lymphocytes and cytokines consisted of the following: purified anti-CD3 (clone UCHT1), anti-CD19 (clone HIB 19), anti-Interferon gamma (clone NIB42), anti-interleukine 2 (clone MQ1-17H12), and Mouse IgG (IgG1 isotypic control) were all supplied by eBiosciences (Rennes, France). Antibodies used for cytokine immunosandwich detection are listed below: polyclonal Anti-Human IL-2 Biotin (1 mgmL?1) and monoclonal Anti-Human IFN-? Biotin (clone 4S.B3; 1 mgmL?1) were also purchased from eBiosciences (Rennes, France). 2.2. Cell Sample In this work, primary T lymphocytes isolated from the.