Collection and Lipidomic Analysis of Murine Knee Synovium and Infrapatellar Fat Pad

Intra-articular soft connective tissues such as synovium and adipose tissue play a crucial role in governing joint homeostasis and disease progression in various forms of arthritis. In the knee, like many synovial joints, adipose tissue forms an integrated anatomic and functional unit with the joint-lining synovium, and the most prominent adipose depot is the infrapatellar fat pad (IFP). With growing evidence that lipid profiles in the synovium-IFP unit shift during progression of joint diseases like osteoarthritis (OA), there is strong impetus for consistent tissue collection approaches and reproducible subsequent lipid characterization. Here, we present a standardized dissection and low-input untargeted lipidomics workflow optimized for mouse knee synovium and IFP, to enable comprehensive lipid profiling. Synovium/IFP from multiple joints are pooled to increase input mass and guarantee robust lipid yield, followed by lipid extraction and high-resolution liquid chromatography-mass spectrometry (LC-MS) acquisition for global, untargeted lipidomic profiling. The analysis workflow encompasses robust feature detection, accurate lipid annotation, data transformation and normalization. These steps enhance comparability across samples, particularly those with low input amounts, while minimizing technical variance and batch effects. Using this approach, we detect a broad spectrum of lipid species spanning the major lipid categories. As expected for untargeted discovery, a subset of non-lipid species is also observed. This protocol provides a practical framework for robust, reproducible lipidomics in murine intra-articular soft tissues to support future disease-specific biomarker and drug target discovery in OA and other joint diseases.