Guava easycyte flow cytometer

Apoptosis assays were performed using an Apoptosis Detection Kit (BD Biosciences). Cells from different treatment groups were trypsinized with 0.25% trypsin, and assays were conducted in accordance with the manufacturer's protocol. In total, 10000 cells were counted in each treatment group using a Guava^® easyCyte flow cytometer (Merck KGaA).
For the analysis of mitochondrial mass, cells were grown in 6‐well plates and treated with the different agents. Cells were trypsinized with 0.25% trypsin and suspended cells were stained with MitoTracker™ Red FM (Invitrogen) (1:10000) for 30 min at 37℃. Cells were then rinsed three times with PBS, and 10,000 cells were counted for each treatment group using a Guava^® easyCyte flow cytometer (Merck KGaA).
The production of ROS was measured by a ROS Assay Kit (Beyotime Biotechnology). In brief, cells were collected and resuspended in DMEM medium after treatment and then incubated with DCFH‐DA for 30 min at 37°C. Cells were then rinsed three times in PBS, and 10,000 cells were for each treatment group using a Guava^® easyCyte flow cytometer (Merck KGaA).

The growth of T. pyriformis and T. thermophila was determined when incubated in Tris buffer and periodically fed with the Brevundimonas SPF441 isolate. Briefly, both ciliates were grown in SPP media as described above to a concentration of 1.0 × 10⁶ cells/ml. The cells were washed twice in 10 mM tris (pH 7.5). Tetrahymena cells were counted using a Guava easyCyte flow cytometer, using the FSC and SSC parameters. The cells were then diluted down to 1.0 × 10³ cells/ml for the co-culture and 1.0 × 10⁴ cells/ml for the control (ciliates alone), in 25 ml of 10 mM tris (pH 7.5) solution. Ciliate cultures were counted before inoculation on day 0 and incubated at 25 °C and 30 °C, for T. pyriformis and T. thermophila respectively. Every other day, starting on day 1 of incubation, 200 μl of 1.000 OD_{600 nm} of Brevundimonas SPF441 isolate culture, washed twice in 10 mM tris solution, was inoculated into the ciliate cultures. Ciliates counts were measured on days 0, 2, and 4 using a Guava easyCyte flow cytometer.