A bioluminescent method based on the reaction of cytosolic Ca2+ with
the genetic probe aequorin in wild type cells was employed as previously
described 12 (link). Briefly, 100 μl of conidia
at a concentration of 2x106 cells/ml, 5 μM coelenterazine (Santa Cruz
Biotechnology) and minimal medium were added to white opaque 96-well plates and
incubated for 6 hours at 26°C, in the dark, without agitation. Luminescence (in
RLU, relative light units) was captured over time on a Bio-Tek Synergy HT
microplate reader. Maximum levels of luminescence, determined in extra wells by
measuring luminescence during 3 mins after the injection of 100 μl of 3M
CaCl2 in 20% ethanol, were used to normalize each experiment
(cytosolic Ca2+ levels (arbitrary units) = experimental RLU values /
total emitted luminescence). Quantification was performed by summing the
normalized values and data are expressed as mean ± SEM. The values from control
DMSO-treated samples were subtracted from staurosporine-treated samples to
obtain the "staurosporine-induced amplitude of response".
the genetic probe aequorin in wild type cells was employed as previously
described 12 (link). Briefly, 100 μl of conidia
at a concentration of 2x106 cells/ml, 5 μM coelenterazine (Santa Cruz
Biotechnology) and minimal medium were added to white opaque 96-well plates and
incubated for 6 hours at 26°C, in the dark, without agitation. Luminescence (in
RLU, relative light units) was captured over time on a Bio-Tek Synergy HT
microplate reader. Maximum levels of luminescence, determined in extra wells by
measuring luminescence during 3 mins after the injection of 100 μl of 3M
CaCl2 in 20% ethanol, were used to normalize each experiment
(cytosolic Ca2+ levels (arbitrary units) = experimental RLU values /
total emitted luminescence). Quantification was performed by summing the
normalized values and data are expressed as mean ± SEM. The values from control
DMSO-treated samples were subtracted from staurosporine-treated samples to
obtain the "staurosporine-induced amplitude of response".
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