Optimized Tissue Preparation Technique in a Chronic Graft-Versus-Host Mouse Model
Abstract
Introduction: Hematopoietic stem cell transplantation (HSCT) is a potentially curative treatment of hematological malignancy or disease. One sequela of HSCT is development of chronic graft-versus-host disease (cGvHD), a serious complication in 30-70% of transplant recipients, and a major cause of death following HSCT. The most common target of cGVHD is the skin, followed closely by oral tissues. Mouse models approximate features of human oral cGvHD and are used to elucidate the pathogenesis of oral cGvHD. Challenges in sample preparation are well-known, resulting in the need to optimize critical laboratory steps in sample analysis. Objective: To test a novel protein extraction method for efficacy in the context of a cGvHD mouse model. Methods: An established mouse model (B10.D2 cells into BALB/c mice) was used to simulate cGvHD conditions. Splenocytes and bone marrow cells from donor mice were transplanted into irradiated recipient mice via retro-orbital injection. Recipient mice were euthanized at specified timepoints post-transplant, and submandibular gland and buccal mucosa tissues were obtained. Tissues were prepared for characterization using a novel liquid nitrogen-cooled mini mortar and pestle technique. Samples were pulverized in microcentrifuge tubes in the mini mortar and combined with appropriate buffers for western blotting (WB) and enzyme-linked immunosorbent assays (ELISA). Protein quantification via bicinchoninic acid (BCA), WB, and ELISA were performed and compared to results from standard extraction techniques. Results: BCA protein quantification showed extraction yields comparable to conventional methods (25.8 g/ml vs 25.6 g/ml for control). WB and ELISA confirmed the presence of proteins and cytokines found in cGvHD. Absence of protein degradation was verified by WB and total protein staining. Conclusion: Protein extraction via the liquid nitrogen-cooled mini mortar technique is an efficient way to prepare mouse oral tissues for analysis.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jun 30, 2020
- Accession Number
- AD1182681
Entities
People
- Eric R Draper
Organizations
- Uniformed Services University of the Health Sciences