Microinjection of Lipid Vesicles into HeLa Cells

Microinjection into HeLa cells was performed as described^{26 (link),27 (link)}. 2 mM lipid (proteo)liposomes, 10 µg/ml DAPI (injection marker) in HB150 were filled in Femtotips (Eppendorf). HeLa cells harvesting 12 mm coverslip was placed into a 35 mm petri dish (Becton Dickinson) filled with pre-warmed culture medium (F12 medium (Invitrogen), supplemented with 10% FCS, 10 mM HEPES (pH7.5) and 100 units/mL each of penicillin and streptomycin). Microinjection was performed using Injectman micromanipulator (Eppendorf) under a Leica DMIL inverted microscope. After microinjection, the cells were incubated for 5 min at 37 °C in the culture medium and then fixed with 4% paraformaldehyde (Sigma-Aldrich) for 10 min followed by immunostaining using antibodies specific for organelles as indicated. To label transferrin-positive endosomes, 5 µg/ml Alexa Fluor 488-Transferrin (final concentrations) were added to the culture medium at the beginning of the injections. After microinjection, the cells were incubated for 5 min at 37 °C and then processed for immunostaining as above. For internalization of Alexa Fluor 488-EGF into endosomes, after HeLa cells were starved in DMEM medium (Lonza) for 3 h, microinjection was performed in 100 ng/ml Alexa Fluor 488-EGF containing injection medium for 5 min and incubated for 5 min at 37 °C in the cell culture medium.