Major histocompatibility complex class II (
MHC II) peptide complexes can associate with
lipid rafts, and this is a prerequisite for their recruitment to the immunological synapse and for efficient T cell stimulation. One of the most often used criterion for raft association is the resistance to extraction by the
detergent Triton X-100 (TX-100) at low temperature. For MHC II, a variety of
detergents have been used under different conditions, leading to variable and often conflicting conclusions about the association of MHC II with
detergent-resistant membranes (DRMs). To clarify whether these inconsistencies were caused by variations in the isolation protocols or reflect different biochemical properties of MHC II
lipid complexes, we used two standardized procedures for the isolation of membranes resistant to
TX-100, 3-[(3-cholamidopropyl)dimethylammonio]-1-
propanesulfonate (
CHAPS), or
Brij 98. Our results suggest that some of the reported variations in the association of MHC II with DRMs are caused by differences in the methods. We also show that in our hands, specific and efficient flotation of MHC II and the MHC II-associated
invariant chain from mouse B-
lymphoma cells was only achieved with
Brij 98, but not with
TX-100 and
CHAPS. We furthermore used DRMs prepared from
hen egg lysozyme-fed B-
lymphoma cells to activate the T cell hybridoma 3A9. In agreement with our biochemical data, T cell activation could only be achieved with
Brij 98- but not with TX-100-resistant membranes. Thus, MHC II and also the
invariant chain belong to a set of
proteins comprising the
T cell receptor,
prominin, and the
prion protein, which reside in membrane environments distinct from conventional
lipid rafts.