Powergen 125 rotor stator homogenizer

The numbers of algae in animals and (where appropriate) in the ASW from the tubs were determined as described previously [51 (link)]. Briefly, animals were frozen and subsequently thawed and homogenized using a PowerGen 125 rotor stator homogenizer (Fisher Scientific). Algal cell numbers were then determined using a Guava easyCyte HT 2 laser flow cytometer with the InCyte v2.7 software (EMD Millipore), which allows highly precise counts (plus/minus ~2% [51 (link)]). ASW containing algae released from anemones was passed through a 27-gauge syringe 10 times in order to disrupt boluses of algae and allow effective counting of single cells with the flow cytometer. Protein concentrations of the homogenates were determined using the BCA assay (see above); this method has precision comparable to that of the Guava [51 (link)].

Caspase activity was determined using the ApoAlert Fluorescent Caspase-3 Kit (Clontech Laboratories, Cat. No. 630215), which should detect the activities of Aiptasia caspases similar to mammalian caspases 3, 7, and (probably) 8 (S1 Fig) [49 (link),50 (link)]. Individual fresh (not previously frozen) anemones were homogenized in 300 μl lysis buffer (from the kit) using a PowerGen 125 rotor stator homogenizer (Fisher Scientific) [51 (link)]. Algal cells [which are not disrupted by the homogenizer [35 (link)]] were removed by centrifugation at 13,000 rpm for 10 min at 4°C in a table-top centrifuge, and the supernatants were frozen for later analysis. For analysis, the extracts were thawed, mixed 1:1 with 2X Reaction Buffer containing dithiothreitol and DMSO, and incubated with the substrate Ac-DEVD-AFC at 37°C for 1 h according to the manufacturer’s instructions. Fluorescence (excitation at 400 nm, emission at 505 nm) readings were then taken using an Infinite Pro spectrophotometer (Tecan, Männedorf, Switzerland). The relative fluorescence units obtained for each sample were normalized to the total protein present in that sample, as determined using the Pierce BCA Protein Assay (Thermo Scientific) according to the manufacturer’s instructions; absorbances were read at 562 nm using the Infinite Pro spectrophotometer.