Cytotoxicity assays provide an in vitro evaluation of the lytic activity of NK and T cells against tumors or transformed cells. However, none of these methods allow the recovery of cells or supernatants after the assay. We standardized a microcytotoxicity test using calcein-acetoxymethyl (calcein-AM) dye that requires very small quantities of cells while maintaining the same sensitivity as the traditional 51 Cr assay. The assay is applicable to resting as well as activated human effector cells and uses different targets such as human cell lines that are adherent or growing in suspension and resistant or sensitive. The most important feature of the method is the possibility of recovering cells and supernatants for additional analyses such as phenotyping and evaluation of soluble factors.Cytotoxicity assays provide an in vitro evaluation of the lytic activity of natural killer (NK) and T cells against tumors or transformed target cells (3,16,25). In in vitro experimental conditions, lytic activity is evaluated using isotopes or dyes either released from dead cells or retained by living ones. The 51 chromium ( 51 Cr) release assay is the most widely used method although it has several significant drawbacks. The major disadvantage results from the use of a radioactive compound, with the related problems of handling and disposal due to the short half-life of the isotope.To overcome these problems, several nonradioactive methods have been developed, but none have yet found a broad acceptance, probably due to a lack of comparability of their results with the results obtained using the 51 Cr release assay, which still remains the most popular. Some alternative methods consist of measuring endogenous or transfected reporter enzymes released in the supernatant by dead targets (1,11,19,24). Other assays using nonradioactive compounds (dimethylthiazol-diphenyl bromide tetrazolium bromide, methylumbelliferyl heptanoate, Alamar blue) evaluate the variations in metabolic activity, which is directly proportional to the number of viable cells (6, 18). Alternative methods using fluorochromes (e.g., europium, D275, rhodamine-123, carboxyfluorescein diacetate, bis-carboxyethyl-carboxyfluorescein) (7,9,12,14,23,26,28) measure the amount of dye released from or remaining in prelabeled target cells (2, 8, 13). The major drawbacks for these methods appear to be the high spontaneous release of the fluorescent dye, the slow specific release of the fluorescein dye, and the low intensity of the fluorescence signal, all of which decrease the overall sensitivity of the test.Among fluorescent dyes, the use of calcein-acetoxymethyl (calcein-AM) in cytotoxicity assays has already been described (13,21,22). This dye has good retention in targets and low pH sensitivity, and there is no stain transfer among cells (21). Acetoxymethyl ester of calcein is a lipid-soluble diester fluorogenic esterase substrate that passively crosses the cell membrane and that is frequently used to stain viable cells. Inside the cells it is converted by intracellular ...

BackgroundThe aim of the study was to characterize synovial cells from OA synovium with low-grade and moderate-grade synovitis and to define the role of synovial macrophages in cell culture.MethodsSynovial tissue explants were analyzed for the expression of typical markers of synovial fibroblasts (SF), synovial macrophages (SM) and endothelial cells. Synovial cells at passage 1 (p.1) and 5 (p.5) were analyzed for different phenotypical markers by flow cytometric analysis, inflammatory factors by multiplex immunoassay, anabolic and degradative factors by qRT-PCR. P.1 and p.5 synovial cells as different cell models were co-cultured with adipose stem cells (ASC) to define SM effects.ResultsSynovial tissue showed a higher percentage of CD68 marker in moderate compared with low-grade synovitis. Isolated synovial cells at p.1 were positive to typical markers of SM (CD14, CD16, CD68, CD80 and CD163) and SF (CD55, CD73, CD90, CD105, CD106), whereas p.5 synovial cells were positive only to SF markers and showed a higher percentage of CD55 and CD106. At p.1 synovial cells released a significantly higher amount of all inflammatory (IL6, CXCL8, CCL2, CCL3, CCL5) and some anabolic (IL10) factors than those of p.5. Moreover, p.1 synovial cells also expressed a higher amount of some degradative factors (MMP13, S100A8, S100A9) than p.5 synovial cells. Co-culture experiments showed that the amount of SM in p.1 synovial cells differently induced or down-modulated some of the inflammatory (IL6, CXCL8, CCL2, CCL3, CCL5) and degradative factors (ADAMTS5, MMP13, S100A8, S100A9).ConclusionsWe found that p.1 (mix of SM and SF) and p.5 (only SF) synovial cells represent two cell models that effectively reproduce the low- or moderate-grade synovitis environment. The presence of SM in culture specifically induces the modulation of the different factors analyzed, confirming that SM are key effector cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-0983-4) contains supplementary material, which is available to authorized users.

Our findings suggest the existence in human chondrocytes of a novel catabolic pathway, primed by chemokines and their receptors, that leads to the breakdown of cartilage matrix components.

These results suggest that IL-17 is an in vitro stimulator of angiogenic factor release, both by its own action and by cooperating with TNF-alpha.

These data suggest that IL-17 could contribute to cartilage breakdown and synovial infiltration in OA by inducing both the release of chemokines by chondrocytes and synovial fibroblasts and, in a less extent, the synthesis of IL-1beta by chondrocytes.