Using TIRF microscopy to analyze stimulated and basal state B-cell MHC II clustering in response to ageing and dietary fish oil

Abstract

This research focused on developing an efficient TIRF microscopy approach to evaluate membrane protein organization. More specifically, the data demonstrate that TIRF microscopy can detect changes in ex vivo B-cell MHC II lateral organization using a monoclonal antibody under differing conditions. MHC II clustering is dependent on the underlying lipid environment and upon LPS stimulation MHC II expression is dramatically increased. Using mice fed a fish oil or control diet for three weeks, or using mice aged for nine months, we imaged splenic B-cell MHC II clustering using TIRF microscopy. We also used LPS to stimulate B-cells from both experimental conditions to determine if either ageing the animals or feeding them fish oil could affect MHC II clustering. We then determined cluster quantity, size, and intensity using the NIH ImageJ software. The data showed that neither a relevant dose of fish oil, nor aging the mice approximately nine months, had an affect on MHC II clustering in the absence of LPS stimulation. However upon LPS stimulation, MHC II clustering dramatically changed in aged mice as well as fish oil fed mice compared to control animals. Taken together, the data establish the TIRF microscopy protocol as a relevant alternative to more costly and time consuming approaches to address membrane protein clustering. Moreover, either ageing the animals or feeding them fish oil significantly affects MHC II clustering upon stimulation with LPS.