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Protocol Steps  >  stimulation step  >  Spore stimuli

Spore stimuli

stimulation step

Protocol: Yeast culture and preparation

In order to test homogeneous yeast populations, pure spore cultures are obtained by using this strain whose sporulation efficiency is 100%. To prepare spores, cells are grown on YPD plates, replicated on SPOIV medium (2% potassium acetate, 0.25% yeast extract, 0,1% glucose) and sporulation is assessed by optical microscopy. Zymolyase (2 mg/ml) is used to digest ascum and liberate spores. [We initially performed experiments to evaluate zymoliase sensitivity of asci for this specific strain and we assessed that 15 minutes incubation was the minimum time needed to damage the ascus, without touching the spore cell wall.] Zymoliase is 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 are 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 is plated on YPD in triplicate. [The spores showed 100% viability.] The procedure used allows exposing DCs to the pure spore culture and to analyze the response to the spores alone, eliminating confounding effects of the presence of the ascus. Although from a mycological point of view the term ascospores might seem more appropriate than spores, we will use the term spores to specifically indicate that the effects we are observing are only due to the spores and not to the ascus.
We observed that after 24 hours of culture in RPMI 1640 (GIBCO-BRL), spores were germinating and the exponentially growing cells were starting to divide. Therefore any analyses performed at later time points would be affected by this altered state, while no interference should be present until 24 hours.

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