Chondrocyte-T Cell Adhesion Assay

For investigation of interaction between chondrocytes with T-lymphocytes an adhesion assay was performed as previously described [14 (link)]. Briefly, primary human chondrocytes were grown to subconfluence in monolayer culture and either left untreated alone or co-cultured with T-lymphocytes (Jurkat cells) (1×10⁶/ml), or were treated with 10ng/ml TNF-β, or 10ng/ml TNF-β and 5μl/ml anti-TNF-β, or 10ng/ml TNF-α, or 10ng/ml TNF-α and 5μl/ml anti-TNF-α and then co-cultured with T-lymphocytes for 8h. Additionally, in another set of experiments, primary human chondrocytes were either co-cultured with T-lymphocytes and/or with 5μM resveratrol and/or 10ng/ml TNF-β and/or 10ng/ml TNF-α for 8h, or were transfected with Sirt1-ASO or Sirt1-SO in the presence of Lipofectin (10μl/ml) for 24h prior to co-treatment with T-lymphocytes and 5μM resveratrol and 10ng/ml TNF-β or 10ng/ml TNF-α for 8h. After 8h of co-culture, non-adherent T cells were carefully removed by gentle washing once with Hank´s balanced salt solution (Biochrom, Germany) and photographed under a light microscope (Zeiss, Jena, Germany). The number of adhered T-lymphocyte colonies on chondrocytes was determined by scoring 10 different microscopic fields. This experiment was repeated three times independently and statistical analysis was done to obtain the final values.