CIL: 50882. In Cell Image Library

The cell surface of exponentially growing Saccharomyces cerevisiae cells was labeled with biotin by incubating the cells with 0,5 mg/ml Sulfo-NHS-LC biotin (#21335, Thermo Fisher scientific) for 20 minutes at room temperature. Cells were grown in YPD, harvested prior saturation of culture, and washed in PBS + 0,5% glucose. Biotinylated cells were labeled with 17.5 ug/ml Streptavidin magnetic beads (#21344, Thermo Fisher scientific) for 1.5 hours followed by 3 x 15 min magnetic sorting with PBS + 0,5% glucose washes in between. Another two rounds of growth, streptavidin-labeling, and sorting was performed. The unbound daughter cells were recovered in YPD for 4 hours before high pressure freezing. Samples were high pressure frozen in a Wohlwend Compact 3 machine, followed by a short freeze substitution protocol in 2% Uranyl acetate and embedded in HM20 resin. Sections of 70 nm thickness were then contrast stained with 2% Uranyl acetate and Reynold's lead citrate. Pictures were aquired in a Tecnai T12 TEM with a Ceta CMOS 16M camera.