Technical Reference #1915

1915.

Regulation of O-glycosylation through Golgi-to-ER relocation of initiation enzymes David J. Gill; Joanne Chia; Jamie Senewiratne; and Frederic Bard, Institute of Molecular and Cell Biology, Journal of Cell Biology, 189(1915), (2010)
Link To Paper

Materials & Methods:
Microinjection SYF and WI38 cells were seeded at desired densities in 35-mm glass-bottom Petri dishes (MatTek) and incubated overnight at 37°C and 10% CO2. The next day media was supplemented with 20 mM Hepes (pH 7.0; 20°C) before cells were microinjected using a 0.5-μm Femtotip-loaded microinjector (model 5242; Eppendorf) equipped with a microscope (Axiovert 35; Carl Zeiss Inc.) fitted with a heated stage. DNA constructs were diluted to 25 ng/μl and centrifuged for 10 min at 10000 g before microinjection into the cell nuclei at 90 hPa for 0.4 s. For co-microinjection DNA constructs were mixed in a 1:1 molar ratio to yield a final concentration of 25 ng/μl. Cells were incubated at 37°C and 10% CO2 for indicated times before either live cell imaging or fixation and staining as outlined above.

Microscopic Technique
Immunofluorescence and Confocal Microscopy

Cell Type(s)
SYF and WI38 Cells