Pierce micro bcatm protein assay kit

For all the cnidarian animals, symbiont cells in the pellets were counted using a BD LSRFortessa^TM Cell Analyzer (BD Biosciences) based on their chlorophyll fluorescence and forward-scatter signals (Supplementary Fig. 26). No obvious change in symbiont cell size or fluorescent intensity has been observed from flow cytometry profiling. Host proteins in the supernatants were quantified using a Pierce Micro BCA^TM Protein Assay Kit (Thermo Fisher) according to the manufacturer’s recommendations. Cell density was determined by normalizing the total cell number to the total host protein content. The normality of cell density data was tested using the Shapiro-Wilk test followed by Levene’s test of homogeneity of variance. Statistical differences among conditions were calculated using two-tailed Welch’s t-tests.

To assess the effects of nutrient supplementation on the key enzymes, we measured the activities of GS and GOGAT in S. pistillata, E. diaphana, and C. andromeda. The same experimental setups as in the symbiont density measurement were followed here, except that only three biological replicates were used for each of the four conditions: control, 10 mM glucose, 250 μM ammonium, and 10 mM glucose plus 250 μM ammonium. After 12 days of treatment, the animals were collected and lysed as described above. The host protein content of each sample was then measured using a Pierce Micro BCA^TM Protein Assay Kit (Thermo Fisher). GS activity was measured using a Glutamine Synthetase Activity Assay Kit (ab284572, Abcam). GOGAT activity was measured using a Glutamate Synthase Microplate Assay Kit (CAK1064, Cohesion Biosciences). One unit of enzyme activity was then defined as the amount of enzyme that produces 1 nmol of ADP (for GS) or decomposes 1 nmol of NADH (for GOGAT) per minute at pH 7.5 at 26 °C. The calculated enzyme activities were then normalized to the corresponding host protein contents.