Semi-quantitation of superoxide
Superoxide (O2-) levels were
quantified according to (Förster, Osmond, & Pogson, 2005) with some
modifications. A volume representing ~12,500 cells of
UWO 241 or SAG 49.72 mid-log phase cultures grown under control
conditions was treated with 20 µL of 1 mM nitroblue tetrazolium (NBT;
Sigma) in the dark for 5 min prior to the stress treatment. Samples were
then filtered onto 25 mm GF/C filters (Whatman, Cat No. 1822-025) and
exposed to short-term stress either LT (5° C) or HL (300 μmol
m-2 s-1) in an AlgaeTron 130 growth
incubator (Photon Systems Instruments, Czech Republic) for up to 1 hr.
Following treatment, filters were immediately immersed in 80% acetone
to remove Chl, and then allowed to dry in a fume hood prior to imaging.
O2- levels were measured
semi-quantitatively by densitometric analyses using the program ImageJ
(http://imagej.nih.gov/ij/).