Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

The overall goal of this experiment is to purify the membranous compartment, where ERAD-like degradation of exogenous antigens is carried out in cross-presentation.This method can help answer key questions in the immunity period, such as the elucidation of cellular compartment or ERAD-like degradation in cross-presentation.The main advantage of this technique is that the purified vesicles contain all molecular operations.That enables ERAD-like degradation of all exogenous antigens.So this method can provide insight into cross-presentation.By dendritic cells, it can also be applied to other types of antigen-presenting cells which show cross-presentation ability.Generally, individuals new to this method will struggle because it is impossible to exclude all non-specific background.A day before purification, split the DC2.4 cells to 0.1 million cells per mL in a tissue culture plate in RPMI buffer.Avoid keeping the cells at a confluent state to increase probability of exogenous antigen incorporation.Before the DC2.4 cells are semi-confluent, add biotinylated ovalbumin, or bOVA, as an exogenous antigen.Then, incubate for two to four hours.Harvest the cells by gentle pipetting from the tissue plate into a new 50 mL conical tube.Centrifuge the cells at 1000 X G for five minutes at 4