Optimization of Methods to Quantify in Situ Staining for UDP-glucose 6-dehydrogenase (UGDH) Enzyme in Human Fibroblast Like Synoviocytes (hFLS) Cultured Under Different Inflammatory Cytokine Conditions

Abstract

UDP-glucose 6-dehydrogenase (UGDH) is an enzyme that catalyzes the conversion of UDP-glucose into UDP-glucuronate. This transformation allows for the biosynthesis of hyaluronan, a glycosaminoglycan found in normal synovial fluid, which is imperative for patients suffering from knee injuries. The purpose of this study was to measure UGDH enzyme activity in human fibroblast like synoviocytes (hFLS), cultured under various inflammatory cytokine conditions. Quantitative histomorphometry methods were optimized using 10x and 20x magnification images of hFLS monolayers from two donors cultured in serum-free (SF) medium or 10% FBS containing medium. The monolayers were submitted to enzyme staining for UGDH, counterstained with Hoechst dye, mounted using Aquamount and images were acquired using inverted epifluorescent microscope. Images were taken of one representative field per well at 20X magnification at a fixed light intensity of 7.4V and exposure time of 0.35 ms. ImageJ software was used to quantify the mean pixel intensity per cell of the UGDH staining in hFLS monolayers (irrespective of cytoplasmic or nuclear localization). This was quantified by dividing the mean pixel intensity derived from the cytoplasmic image set to a threshold of 185 by the number of nuclei manually counted. hFLS cultured in 10% FBS medium yielded a 4-fold higher UGDH enzyme staining intensity per cell in comparison to hFLS cultured in serum-free medium (p=0.0023). In addition, there was a positive correlation between cell density and staining intensity.