50 ml tube

The donated stool is processed within two hours of defecation. The stool samples were stored on room temperature until the begin of the processing. Reusable tools are sterilized with ethylene oxide. After adding 200 mL of sterile physiological saline solution (0.9% sodium chloride) to 60 g of the sample, the stool is homogenized with a household mixer (AEG HM 250). The initial solution is filtered with a household pasta sieve to eliminate debris. A secondary screening is carried out with another sieve with a finer mesh. Depending on the consistency of the faecal matter, the resulting volume is approximately 220 mL. This volume is then placed in 50 mL tubes (Sarstedt Inc., Nümbrecht, Germany) and centrifuged for 10 minutes at 827 g (MPW-380R, Poland) to eliminate the smaller debris. The slurry is discarded, and the supernatant is handled in 100 mL portions. With these steps, approximately 120 mL of macroscopically homogenous solution can be made from 60 g of initial stool sample. This procedure is carried out under laminar airflow in a dedicated cabinet on room temperature.

Ovaries were obtained from non-pregnant domestic cats after routine ovariectomy at animal shelter of Berlin. The ovariectomia were not related to the purpose of the experiment. Ovaries were transported to the laboratory in HEPES-MEM medium, supplemented with 3 g/L BSA and 1x Antibiotic Antimycotic Solution in 50 mL tubes (Sarstedt AG & Co. KG, Nümbrecht Germany). Upon arrival, ovaries were isolated from surrounding tissues and washed twice in Dulbecco PBS (DPBS), and checked for the presence of CL indicating a non-pregnant luteal cycle. CL were isolated and washed in fresh DPBS. Half of one CL per animal was fixed in Bouin solution and used for identifying the stage of the luteal life cycle as described by Amelkina (25 (link)). For the purpose of the experiments, luteal cell cultures from 17 queens were made, of which 9 were defined as development/maintenance stage. Due to the small size of cat CL, 0.3 × 10⁶ SLC and 0.1 × 10⁶ on average LLC were able to be isolated from one CL. Thus, a full experimental trial including gene expression analysis on both cell types obtained from the same domestic cat was only possible in six cases. The data from these selected experiments (n = 3 for experiment A; n = 3 for experiment B) were compiled for statistical analysis. All other experiments contributed to the microscopic and steroidogenic characterization (see below) of SCL and LLC.

Giardia intestinalis isolate WB, clone C6 (ATCC 30957), was used in this study. For validation by standard fluorescence microscopy, a modified Giardia line constitutively expressing mNeonGreen was generated (described below). Giardia trophozoites were grown at 37°C in 10-ml flat plastic tubes (Thermo Fisher Nunc, MA, USA) or 50-ml tubes (Sarstedt, Germany) filled with TYDK medium (also known as modified TYI-S-33 or Keister's medium) (79 (link)), supplemented with 10% heat-inactivated bovine serum (Gibco, Thermo Fisher MA, United States). All materials used in the TYDK medium were purchased from Sigma-Aldrich (MO, USA) unless otherwise stated. For IEC monolayer infections, Giardia trophozoites were grown until approximately 70% confluence and washed once with TYDK to remove dead cells. Further, trophozoites were incubated on ice (12 min), counted, and pelleted by centrifugation (800 × g, 10 min, 4°C). Cells were washed once in 1 ml DMEM-F12 (Thermo Fisher [Gibco]), centrifuged, and diluted to 2 × 10⁷ or 4 × 10⁷ trophozoites/ml using DMEM-F12, of which 10 μl was used for infection, resulting in 2 × 10⁵ to 4 × 10⁵ trophozoites per monolayer.

Spleens were placed on a Petri dish containing RPMI 1640 and cut into small pieces. The resulting tissue was passed into a 70 μm cell strainer (BD, 352350) and broken up in a circular motion with the ribbed top of a syringe’s plunger. RPMI 1640 was then passed through the strainer. The disintegrated tissue was transferred to 50 mL tubes (Sarstedt, Barcelona, Spain, 62.547.254) and centrifuged at 100 g for 10 min. The supernatant containing the cell suspension was then collected and centrifuged again at 300 g for 10 min. The resulting supernatant was removed, and the pellet was resuspended in 10 mL phosphate-buffered saline (PBS) with 1% FBS to count the number of cells under the microscope, after which it was re-centrifuged at 300 g for 5 min. Only 500,000 cells per sample were used. Each sample was then resuspended in 50 µL of blocking buffer before staining for flow cytometry.

Whole-lung lavage (WLL) fluid was collected from consenting patients undergoing WLL at our hospital as a trial treatment for silicosis, as reported earlier [21 (link)]. The lavage fluid was collected sequentially into 3-litre containers and returned to our laboratory on-site for processing. The fluid was decanted into 50 mL tubes (Sarstedt, Mawson Lakes, Australia) for centrifugation (800× g, 8 min) and resuspended in 20 mLs of RPMI 1640 (Gibco, Thermo Fisher Scientific, Seventeen Mile Rocks, Australia) supplemented with 2% heat-inactivated fetal calf serum (FCS) (Gibco) and penicillin/streptomycin/glutamine (Gibco), then combined with an equal volume of FCS containing 15% dimethyl sulfoxide (DMSO)(Sigma, Merck life Science, Bayswater, Australia). Cells were then cryopreserved in 40 × 1 mL aliquots in liquid nitrogen until later use.

Experiments were conducted under well-mixed conditions with 5 mL medium in 50 mL tubes (Sarstedt). Exponential growth rates (S1 Fig and S2C Fig) were measured in a Tecan Infinite M200 reader on 100 μL cultures with 50 μL mineral oil (Sigma) in 96-well plates, after 100-fold dilution from stationary phase cultures.