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/).