metaview image analyzing system version 4.50  (universal imaging inc)

Journal: Scientific Reports

Article Title: Omega-3 fatty acid-rich fish oil supplementation prevents rosiglitazone-induced osteopenia in aging C57BL/6 mice and in vitro studies

doi: 10.1038/s41598-021-89827-8

Figure Lengend Snippet: Effect of RSG with or without omega-3 FAs on osteoblastogenesis and adipogenesis in C3H10T1/2 cells. C3H10T1/2 cells were plated in a 48-well plate and cultured in α-MEM medium with 10% FBS supplemented with recombinant human (rh) BMP-2 (Biovision, USA) and cultured for 7 days in the presence of RSG 1 µM with or without EPA 10 µM and/or DHA 10 µM. Representative photographs (20×) of C3H10T1/2 cells stained for alkaline phosphatase (elongated cells) ( a ). Optical density measured by Metaview Image Analysis (Olympus Inc. USA) in RSG treated cells with and without FO (EPA and DHA) in the presence of BMP-2 ( b ). Cells were lysed with lysis buffer and quantified for protein content. Alkaline phosphatase (ALP) activity was measured by using SensoLyte pNPP ALP Assay by mixing cell lysates with pNPP reaction mixture and incubated for 30 min. ALP activity in each sample was determined by measuring OD405 and compared to an ALP standard curve ( c ). Cells were then stained with Oil red O using fast red AL salt as a substrate. Representative photographs (20x) of C3H10T1/2 cells stained for Oil red O (round cells) ( d ). Optical density measured by Metaview Image Analysis (Olympus Inc. USA) in RSG-treated cells with and without FO (EPA and DHA) supplementation ( e ). Each bar represents the mean ± SEM of 6 wells per group. Value with different superscripts is significantly different at p < 0.05 by Newman–Keuls’ one-way ANOVA with multiple comparison tests. ALP Alkaline phosphatase, RSG rosiglitazone, EPA eicosapentaenoic acid, DHA docosahexaenoic acid, MEM minimum essential medium, BMP-2 bone morphogenetic protein 2.

Article Snippet: Wells were then carefully rinsed with PBS/EDTA 20 mM and the ALP-stained optical density (OD) intensity was analyzed by histomorphometry using the Metaview Image Analysis System (Olympus Inc. USA) .

Techniques: Cell Culture, Recombinant, Staining, Lysis, Activity Assay, ALP Assay, Incubation

Journal: Scientific Reports

Article Title: Omega-3 fatty acid-rich fish oil supplementation prevents rosiglitazone-induced osteopenia in aging C57BL/6 mice and in vitro studies

doi: 10.1038/s41598-021-89827-8

Figure Lengend Snippet: Effect of RSG with or without omega-3 FAs on osteoclastogenesis in RAW 264.7 cells. RAW 264.7 cells were cultured with RANKL 50 ng/mL in the presence of RSG 1 µM with or without EPA 10 µM and/or DHA 10 µM for 5 days. At the end of the cultures, cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP). The number of TRAP-positive multinucleated cells (MNCs) (more than 3 nuclei) was counted as osteoclast-like cells in each well. Representative photographs (20×) of TRAP-positive MNCs ( a ). Optical Density (OD) intensity was determined by histomorphometry using the Metaview Image Analysis System software for the TRAP-positive area ( b ). Each bar represents the mean ± SEM of two independent quadruplicate cultures. Value with different superscripts is significantly different at p < 0.05 by Newman–Keuls one-way ANOVA with multiple comparison test. RSG rosiglitazone, EPA Eicosapentaenoic acid, DHA Docosahexaenoic acid, RANKL Receptor activator of nuclear factor kappa-Β ligand.

Article Snippet: Wells were then carefully rinsed with PBS/EDTA 20 mM and the ALP-stained optical density (OD) intensity was analyzed by histomorphometry using the Metaview Image Analysis System (Olympus Inc. USA) .

Techniques: Cell Culture, Staining, Software