Purificación del compartimento de la membrana de retículo endoplasmático asociado degradación de antígenos exógenos en presentación cruzada

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