B rker chamber

The spleens were rubbed through a nylon sieve (70 μm, BD Falcon, Corning, NY, USA) and washed with PBS. The resulting cell suspensions were centrifuged for 10 min at 350× g (4 °C). The splenocytes were resuspended in 5 mL of red blood cell lysis buffer and kept for 5 min on ice before being washed with PBS and centrifuged at 350× g for 10 min at 4 °C. To obtain mouse CD4⁺ cells, magnetic separation was performed using a CD4⁺ T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer’s protocol. Cell number and viability were determined using trypan blue (Sigma-Aldrich, USA) staining and counting in a Bürker chamber (Sigma-Aldrich, St. Louis, MO, USA) under a light microscope. Th17 cells were obtained from collected CD4⁺ T cells using the Flow Cellect^™ Mouse Th17 Differentiation Tool Kit (Millipore Merck, Darmstadt, Germany) according to the manufacturer’s protocol. Detailed assay instructions certify to gain complete Th17 cell generation in 6 days. After seeding 2 × 10⁵ CD4⁺ cells on day 1 into each well of the kit’s plate, on day 6 the total number of differentiated cells were approximately 1–5 × 10⁶. All cell types were stained with trypan blue solution and counted in a Bürker chamber under a light microscope.

4T1 cells were cultured in the logarithmic phase to 70% confluency on a tissue culture Petri dish. The supernatant was aspirated, and the cells were washed with 5 mL of PBS (Merck) and detached using 3 mL of Accutase (Thermo Fisher Scientific) at room temperature for 5 min. The cells were harvested by adding 7 mL of PBS and then counted using a Bürker chamber and trypan blue excision dye (Merck). In total, 2 × 10⁵ 4T1 cells were incubated with plasma and then 5× diluted in 100 µL of IFB at 4 °C for 1 h. The cells were washed with 1 mL of IFB and centrifuged at 1500 rpm for 5 min. The cells were resuspended in 100 µL of IFB containing goat-anti-mouse anti-IgG Alexa Fluor^TM 488 (Thermo Fisher Scientific, A-11001 400× diluted), and then incubated at 4 °C for 30 min. The cells were washed with 1 mL of IFB and then centrifuged at 1500 rpm for 5 min. The cells were resuspended in 300 µL of IFB, and 10 µg/mL of propidium iodide was added (3 µL from 1 mg/mL stock) right before acquisition to gate on viable cells. In total, 10,000 events per sample were detected using an FACSCalibur cytofluorimeter, and data analyses were performed using the CellQuest^TM software.

Algae found in water samples after the 9-day experiment were identified mostly to the genus level in accordance with professional manuals [43 ,44 ]. The abundance of bacterial and algal cells was estimated by direct microscopic counting (Zeiss Primo Star, Monument, PA, USA) of cells in the Bürker chamber (Merck Eurolab, Stockholm, Sweden). This is a simple manual method for enumeration of average cell number by counting cells in squares; each square for bacteria counting has an area of 1/400 mm² and sample volume of 1/4,000,000 mL, whereas for algal cells, the area was 1/25 mm² and the sample volume was 1/250,000 mL. For each sample, counting was performed in triplicates and in 20 squares of the counting grid each time.