Paraformaldehyde-Induced Vesiculation Preserves Native Membrane Physiological States Critical for T-Cell Activation

Plasma membrane vesicles (PMVs) are powerful model systems for studying plasma membrane biophysics, but their production typically relies on chemicals, such as dithiothreitol (DTT) and paraformaldehyde (PFA).DTT is detrimental to protein structure and can alter membrane composition, where as PFA is a well-established fixative that is widely used in biological studies. Taking this into consideration, we introduced a DTT-free method for generating PMVs using PFA alone. Using flow cytometry and fluorescence microscopy, we validated its robustness across multiple cell types. Measurements with the environment-sensitive probe C-Laurdan show that PMVs produced with PFA plus DTT display higher lipid packing compared to those generated with PFA alone, indicating an effect of DTT on membrane order. Furthermore, fluorescence correlation spectroscopy (FCS) measurements reveal reduced mobility of the immune receptor cluster of differentiation 1d (CD1d) on DTT-treated PMVs. During the evaluation of PMVs in coculture experiments, we further identified residual PFA as the source of cellular toxicity, which was successfully eliminated by dialysis. Finally, we demonstrate the biological applicability of the resulting PMVs by assessing T-cell activation, showing that they preserve key physiological properties of the source cells. Overall, our findings suggest that PMVs generated using PFA alone more faithfully preserve the native properties of the source cell plasma membrane than those produced with the conventional PFA plus DTT protocol. Combined with the effective removal of residual PFA by dialysis, this approach expands the potential of PMVs as physiologically relevant model systems for biological and biomedical research.