UltrAuFoil on gold 200 mesh R2/2 grids (Quantifoil) were subjected to micropatterning44 (link),45 (link) using the Primo module from Alveole, mounted on a Leica DMi8 microscope, following the manufacturer procedure. Briefly, the grids were coated with polylysine (100 μg/ml, 30 min) followed by mPEG-SVA (100 mg/ml, 1 h) and PLPP gel (1 h) prior to exposure to UV (50 mJ/mm2) to create circular patterns of 40 μm diameter. Then the grids were profusely rinsed with PBS before incubation with fibrinogen couple to Alexa 633 (Thermo Fisher). Micropatterned grids where then stored in Hank’s Balanced Salt Solution (HBSS) (Gibco).
HeLa cells (CCL-2, ATCC, Manassas, VA, USA) were grown in Dulbecco’s Minimal Essential Media (DMEM) (Gibco) with high glucose and non-essential amino acids complemented with 10% FBS, 1% glutamine and gentamycin (25 μm/ml). Cells were seeded on micropattern grids for 2 h before washing. Infection with Chlamydia trachomatis LGV02 were performed as previously described46 (link). 24 h post infection cells were plunge-frozen using the Vitrobot (Thermo Fisher) offsetting the blotting pad to favour back blotting. Just prior to plunge-freezing, the cells media was replaced with the complete media with 10% glycerol (v/v). Vitrified grids were then clipped (Thermo Fisher) into autogrids (Thermo Fisher) and subsequently stored under liquid nitrogen.
Plunge-frozen S. cerevisiae (Yeast) samples were prepared as previously described43 .
HeLa cells (CCL-2, ATCC, Manassas, VA, USA) were grown in Dulbecco’s Minimal Essential Media (DMEM) (Gibco) with high glucose and non-essential amino acids complemented with 10% FBS, 1% glutamine and gentamycin (25 μm/ml). Cells were seeded on micropattern grids for 2 h before washing. Infection with Chlamydia trachomatis LGV02 were performed as previously described46 (link). 24 h post infection cells were plunge-frozen using the Vitrobot (Thermo Fisher) offsetting the blotting pad to favour back blotting. Just prior to plunge-freezing, the cells media was replaced with the complete media with 10% glycerol (v/v). Vitrified grids were then clipped (Thermo Fisher) into autogrids (Thermo Fisher) and subsequently stored under liquid nitrogen.
Plunge-frozen S. cerevisiae (Yeast) samples were prepared as previously described43 .
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