Spore cultures

In order to test homogeneous yeast populations, pure spore cultures were obtained by using this strain whose sporulation efficiency is 100%. To prepare spores, cells were grown on YPD plates, replicated on SPOIV medium (2% potassium acetate, 0.25% yeast extract, 0,1% glucose). Zymolyase (2 mg/ml) was used to digest ascum and liberate spores. Zymoliase was inactivated by heat (65°C for 2 minutes) and washed away carefully together with the remainings of the empty ascus, by resuspending twice the spore culture in 500 µl of distilled water centrifuging and discarding the supernatant; the spores were then resuspended to a concentration of 108 cells/ml. In order to exclude damage on the spore cell wall or effects on spore viability 100 µl of a culture of 1000 spores per ml was plated on YPD in triplicate, the spores showed 100% viability.

The spores were then biotinylated, labeled and - after treatment of DCs - detected using APC-labeled streptavidin and analyzed by flow cytometry.

created over 15 years ago (21 January 2010)    last modified over 13 years ago (28 September 2011)   [ RDF ]   [ RelFinder ]