The amount of NP internalization was determined using flow cytometer (BD FACSCalibur equipped with 488 and 633 nm lasers) in the FL2 channel.
Note also that raising the temperature of the cells from 4 to 37ºC induces a small amount of HER2 internalization [ 15].
The level of cytokine production strongly correlates with the amount of NOMV internalization implying that receptor engagement within the DC is likely to be critical.
That this was a specific event was evident by the notion that only a small amount of (constitutive) internalization happened when vehicle (DMSO) was present.
This would indicate that a significant amount of cellular internalization of 2 is an active process although the low intracellular luminescence in cells incubated at 4 °C does suggest that a background level of passive diffusion also occurs.
This might also explain why, in our ex vivo experiments where neurons had been exposed to morphine for 3 days, the PKC inhibitor did not completely reverse MOPr desensitization, and why a small, but significant amount of receptor internalization was induced by morphine in HEK-293 cells (Johnson et al., 2006) and striatal neurons (Haberstock-Debic et al., 2005).
After binding, the cells were shifted back to 37°C and the transferrin internalization chased at different time points up to 15 min. The amount of internalized ligands was measured by FACS analysis (FacsCalibur, Becton&Dickinson).
To further confirm that treatment with commercial GluA2 antibodies plus secondary antibodies increased net internalization of AMPARs while decreasing synaptic AMPARs, as observed with patient antibodies, we examined the amount of internalized AMPARs.
Although electroporation allows high internalization efficiencies to be achieved, the amount of non-internalized fluorescent molecules that remain in cell suspension after electroporation is typically much higher than the amount of internalized molecules.
GRAF1 depletion resulted in a 50%60%% reduction of total dextran endocytosis, similar to that observed upon depletion of AP2, the major Clathrin adaptor at the plasma membrane, suggesting that GRAF1-mediated endocytosis and Clathrin-mediated endocytosis account for roughly equal amounts of volume internalization in these cells.
