![kbasic function of capillaries kbasic function of capillaries](https://images.squarespace-cdn.com/content/v1/5f591342171ae90d7ca5bc4e/1612550985914-1SZC80P4FQY98T53IDB8/Tofu+-+Made+with+PosterMyWall.jpg)
The specific fluorescence index (SFI) was calculated as the ratio of the mean fluorescence obtained with the specific antibody and the isotype control. Cells (∼10,000) were measured using flow cytometry (FacsCalibur Flow Cytometer BD Biosciences, Heidelberg, Germany). After washing in flow cytometry buffer the cells were incubated for 30 minutes at 4☌ in FITC-conjugated rabbit anti-mouse IgG (Sigma-Aldrich). Isotype controls were incubated with 1 μg/mL nonspecific mouse IgG (Sigma-Aldrich). Cells were washed in cold PBS, incubated for 20 minutes in 10% rabbit serum in flow cytometry buffer (PBS/1% BSA/0.01% sodium azide) and incubating with 1 μg/mL mouse monoclonal antibody against MMP-3 (Ab-5) or TIMP-2 (Ab-2 Oncogene) at 4☌ for 60 minutes. After centrifugation, the cells were permeabilized by resuspension (10 6 cells per tube) in 75% ethanol for 10 minutes at 4☌. In brief, fibroblasts were rinsed in cold PBS, incubated for 3 minutes in trypsin at 37☌ and harvested into complete medium containing 10% FCS. 30 Both untreated and latanoprost-treated cells were used. Tenon fibroblasts may be the target cells for attempts to influence the tissue levels of MMPs and TIMPs in the context of conjunctival wound healing after glaucoma surgery.įlow cytometry analysis was performed as previously described. The levels of MMP-3 and TIMP-2 increase after treatment with latanoprost. Tenon fibroblasts contain the ability on the mRNA level to synthesize all enzymes of the MMP and TIMP family that are related to remodeling of the extracellular matrix. The upregulation of MMP-3 and TIMP-2 was confirmed in vivo.Ĭonclusions. An upregulation of MMP-3 and TIMP-2 after latanoprost treatment of the fibroblasts was shown and found to occur on the mRNA and the protein levels. With the real-time RT-PCR technique, MMP-1, -2, -3, -7, -9, and -14 and TIMP-1 and -2 were detected. In all assays, both MMP-3 and TIMP-2 were detected. To investigate the changes in vivo, conjunctival specimens of rabbits treated with latanoprost eye drops were examined by immunohistochemistry. Assays performed included RT-PCR, real-time RT-PCR, immunocytochemistry, Western blot analysis, flow cytometry, and zymography. Untreated and latanoprost-treated human Tenon fibroblasts were examined for the presence of MMPs and TIMPs on the mRNA and protein levels. This aspect is important to further the understanding of and possibly to influence the scarring process of filtering blebs after glaucoma surgery. These enzymes are essential for the control of tissue remodeling in the context of wound repair. To investigate the presence and the possible role of different matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in Tenon capsule fibroblasts.