Cyan adp flow cytometer

Spleen ex vivo interferon-gamma (IFN-γ) ELISpot was performed as described previously [19] (link). To measure vaccine antigen specific responses, cells were re-stimulated for 18–20 h with peptides (Supplementary Table 1) at a final concentration of 1 μg/mL. Spot forming cells (SFC) were measured using an automated ELISpot reader system (AID). Flow cytometry on peripheral blood mononuclear cells (PBMC) was performed as described previously [37] (link), except that peptide re-stimulation was with 1 μg/mL Pb9. All fluorophore-conjugated antibodies were obtained from eBiosciences. Data were acquired on a CyAn-ADP flow cytometer (Dako) and analysed using FlowJo (Treestar). The program SPICE was used to generate graphical representations of functional T cell responses using background-deducted data [31] (link). IgG endpoint ELISA was performed as described previously [6,15] except plates were coated with recombinant GFP protein (Millipore, UK) at 1 μg/mL.

Neuro-2a cells were seeded 24 h prior to transfection into 24-well plates at a density of 50,000 cells/well. Culture medium was replaced with 400 μL fresh growth medium 24 h after seeding the cells. pDNA polyplexes formed at N/P ratio 12 in 100 μL HBG and containing 1 μg pCMVLuc (20% of Cy5-labeled pCMVLuc) were added to each well and incubated at 37 °C for 45 min. Subsequently, cells were washed once with 500 µL PBS containing 1000 IU heparin for 15 min on ice to remove any polyplexes sticking to the cell surface and again washed once with 500 μL PBS only. Cells were detached with trypsin/EDTA and resuspended in PBS with 10% FBS. All experiments were performed in triplicate. Cellular internalization of the polyplexes was measured by the excitation of Cy5 at 635 nm and detection of emission at 665 nm. DAPI staining was used to discriminate between viable and dead cells. Cells were properly gated by forward/sideward scatter and pulse width for exclusion of doublets. Data were recorded by Cyan ADP flow Cytometer (Dako, Hamburg, Germany) using Summit acquisition software (Summit, Jamesville, NY, USA) and analyzed by FlowJo 7.6.5 flow cytometric analysis software.

Prior to staining, cells were blocked with CD16/32 (Fc Block; BD). For surface staining, antibodies against the surface markers CD4, CD8, CD3, CD69, CD11b, CD11c, MHC class II (MHC-II), CD27, and DX5 (BD) were added at a 1:100 dilution for 30 min on ice. Gating for lymphocytes was determined by back gating on CD3/CD8 double-positive cells. For the detection of intracellular cytokines, cells were incubated with 50 ng/ml phorbol myristate acetate, 500 ng/ml ionomycin, and 10 μg/ml brefeldin A for 4 h at 37°C. Samples were permeabilized with 0.5% saponin in phosphate-buffered saline (PBS) for 10 min. Anticytokine antibodies (anti-IFN-γ, anti-IL-4; BD) or isotype controls were added for a further 20 min at room temperature. Cells were analyzed on a CyAn ADP flow cytometer (Dako), with data on at least 50,000 events being collected.

To quantify changes in mitochondrial membrane potential cells were labeled with 50 nM of the mitochondrial membrane potential-sensitive fluorescent dye TMRE (Invitrogene) for 30 mi a 37 °C, analyzed by a Cyan-ADP Flow Cytometer (DAKOCytomation) and quantified using Summit software.

DSS-induced chronic colitis was initiated by multi-cycle administration of DSS drinking water [27 (link)]. Female mice of eight weeks of age received 2.3% (w/v) DSS (40,000–50,000 MW; MP Biomedicals, Irvine, CA, USA) in drinking water on days 1–5, 8–12, 15–19, and 22–26. Mice were injected with PBS, ES-62 (2 μg/mouse), SMA 11a or SMA 12b (1 μg/mouse each) three times per week starting on day 1 of DSS treatment. Mice were checked daily for the development of colitis by monitoring body weight, gross rectal bleeding, and stool consistency, and were sacrificed at day 29. The cecum and colon were removed, tissues were fixed in 10% formalin in PBS, paraffin-embedded, and cross sections were stained with haematoxylin and eosin (H&E). Lamina propria mononuclear cells (LPMC) were isolated from the large intestine as previously described [28 (link)]. Single-cell suspensions of LPMC were stained with anti-CD4 antibodies for flow cytometry. To determine the percentage of cells expressing surface markers and the intensity of expression, samples were examined using a CyAn™ ADP flow cytometer (Dako Cytomation, Carpinteria, CA, USA) and analysed with the FlowJo (TreeStar, Ashland, OR, USA). Percentages of CD4⁺ cells were analysed on the gated lymphocyte population.

Synovial fluid was incubated with 1000 U/mL endotoxin-free hyaluronidase (Wockhardt UK) at 37°C for 15 min. Mononuclear cells were isolated from synovial fluid and peripheral blood using density gradient centrifugation. Cells were stained with mouse monoclonal antibodies against CD19 (Biolegend), CD20 (Invitrogen), CD27 (BD Pharmingen), IgD (eBioscience), CD11c (Biolegend), RANKL (eBioscience), FcRL4 (Biolegend), CD95 (BD Pharmingen), CD21 (eBioscience), CD80 (BD Biosciences), CD86 (Biolegend), CCR1 (R&D Systems) and CCR5 (BD Biosciences). Isotype, concentration, species and label-matched control antibodies were used. Data were acquired using a Dako Cyan ADP flow cytometer and analysed using SUMMIT software. Synovial fluid mononuclear cells were stained with antibodies against CD19 (Immunotools) and FcRL4 (Biolegend) and sorted using a MoFlo cell sorter (Dako). Sorted populations had a purity of >95%. Mononuclear cells were also isolated from mechanically dissociated synovial tissue as previously described14 (link) from RA patients undergoing joint-replacement surgery which were assessed by flow cytometry for FcRL4 and CD19.