It should be taken into account, too, that the light path in Wnt inhibition typical measuring chambers (usually 1–2 cm) is much smaller than that in the culture vessel (5–10 cm), so that the light intensity reaching every single cell is selleck higher due to less self-shading of the cells. The use of O2 electrodes of the Clark type is a common technology which will not be
explained here in detail. It should be noted, however, that Clark-type electrodes can easily be converted to H2 electrodes just by applying a different potential. Details of assembling and using these electrodes are given in references Wang (1980), Kuroda et al. (1991), and Takeshita et al. (1993). An easy method to analyze in vivo H2-production rates of illuminated C. reinhardtii cells without a H2 electrode will
be described here. This technique is suitable to determine the real H2-evolution rate of the cells, which can be only roughly concluded from the accumulation of H2 in the gas phase of the incubation LCZ696 flask or in a gas trap (see below). For this purpose, a 2-ml sample of the main culture is taken with a syringe by piercing through the septum and gently injected through the rubber seal of an 8–10 ml headspace bottle as described above, which has been gassed with Ar before. The little vessels are then placed in the light. The cell suspension has to be rocked or stirred so that the cells do not settle. A shaking water bath made from plexiglas standing on top a light source is optimal. After 10 min, a volume of the gas phase is analyzed with a gas chromatograph to determine H2 concentration. Then, the cells are incubated for 1 h, and H2 is detected again. The difference of the H2 concentration in the beginning and after 60 min is the amount of H2 that has been produced by the cells. It should be noted that the 10 min of pre-incubation is applied to let the cells adapt to the system, which will differ from the incubation conditions of the main culture in some aspects. Furthermore, during the
transfer of the cells, some air (i.e., O2) might have entered the cell suspension, and the cells might have been shaded to some extent. Non-specific serine/threonine protein kinase During the pre-incubation, the algae will stabilize their H2 metabolism. The first analysis of the H2 concentration after the 10 min duration is important to take into account the H2 which has been produced during this pre-incubation phase and the gas which was introduced into the reaction vessel by the algal suspension. In the active H2-producing phase of S-deprived C. reinhardtii cultures, significant amounts of H2 are dissolved in the medium of the cells. The above point should also be kept in mind when carrying out in vitro hydrogenase activity assays with S-depleted algae.