GPMV Isolation and Preparation.

RBL cells were cultured in medium containing 60% Eagle’s minimum essential medium, 30% RPMI, 10% fetal calf serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in humidified 5% CO₂. GPMVs were isolated from RBLs as described previously (12, 51). Briefly, GPMV formation was induced by 25 mM paraformaldehyde + 2 mM DTT in isotonic GPMV buffer containing NaCl, 10 mM Hepes, and 2 mM CaCl₂, pH 7.4. All GPMVs from one 60-mm cultured dish were combined into a 1.5-mL microcentrifuge tube and concentrated into a loose pellet by centrifugation at 20,000 × g for 20 min. The pellet was resuspended by gentle pipetting in 20 μL of GPMV buffer immediately prior to cryopreservation. Small vesicles present in this preparation were abundant by cryo-EM. These may represent submicroscopic vesicles formed in the same manner as micrometer-scale GPMVs or fragmentation of larger GPMVs into smaller ones during preparation. Both possibilities could conceivably lead to differences in composition or properties of GPMVs. We also note that these GPMVs do not retain all features of native PMs. While their composition is likely to be representative of the PM (47, 75), the phospholipid asymmetry, cytoskeletal interactions, and energy-dependent processes of live cell PMs are lost (46, 51).