**2. Materials and Methods**

#### *2.1. Blastocystis Culture*

*Blastocystis* ST7 cultures were grown axenically in 8 mL of Iscove's Modified Dulbecco's Medium (IMDM) (Gibco-Catalogue no 12,200,069 Thermo Fisher scientific) with 10% heat-inactivated horse serum (HIHS) (Gibco-Catalogue no 26,050,088 Thermo Fisher scientific). All cultures were passaged every 3–4 days depending on their growth rate and were subsequently expanded. All cultures were incubated at 37 ◦C in 95% CO2 and 5% O2. The gas concentration was maintained by a gas pack (BD-Catalogue no 261205) in an anaerobic chamber (Oxoid-Product code 10,107,992 Fisher scientific). Cell counts were achieved manually using a Neubauer haemocytometer (Brand-Catalogue no 717810).

#### *2.2. Cell lysis and Metabolite Extraction*

*Blastocystis* cultures intended for metabolite extraction were pooled in a 50 mL tube and centrifuged at 1000× *g* for 5 min at 4 ◦C and the supernatant was discarded. Resulting pellets were re-suspended in 5 mL of Locke's solution and given 2× washes with Stone's modification of Locke's solution (ATCC medium 1671), which was removed by a subsequent centrifugation at 1000× *g* for 5 min at 4 ◦C. The washed pellets were snap frozen in liquid nitrogen and stored at −80 ◦C.

Three steps were implemented for each experiment to determine the optimum extraction protocol and were each repeated four times. The conditions of each of the 4 experiments are shown in Table 1.

**Table 1.** Conditions of each experiment used to determine the best lysis method, incubation temperature and extraction solvent.


Step 1: Three cell cultures were thawed, resuspended in 5 mL of Lockes' solution and then homogenised by vortexing for 30 s. These were then divided into two equal weight batches for parallel analysis. Each batch was centrifuged at 1000× *g* for 5 min at 4 ◦C, after which the supernatant was removed.

Step 2: The two batches were added to one of two different solvents: either 4 mL of ethanol:water (3:1) or 4 mL of methanol:water (1:1). The two different solvent batches were further processed at either −20 ◦C, room temperature (RT) or 60 ◦C (with samples for each solvent at each of the three temperatures). Each batch was then disrupted using one of two methods; either sonication in 3 × 30 s bursts or bead bashing by vortexing with 200 mg of 0.4 mm glass beads for 30 s followed by a 3-min incubation at either −20 ◦C, RT or 60 ◦C, then followed by vortexing for a further 30 s.

Step 3: Resulting solutions were then divided into 4 × 1 mL aliquots and centrifuged for 15 min at 4 ◦C at 10,000× *g*. The supernatants were decanted into fresh tubes and lyophilised.
