Rpmi 1640 cell culture medium

To further study the mechanisms, a monocyte cell line (U937) treated with plasma of KD or healthy control was applied to investigate the expressions of CASP1, 4, and 5 in regulating KD in vitro. The human macrophage cell line U937 cells (ATCC and CRL-1593) were maintained in a RPMI1640 cell culture medium (Thermo Fisher Scientific) with 10% FBS at 37 °C, 5% CO₂. U937 monocytes were differentiated to macrophage cells by adding phorbol 12-mystrate 13-acetate (PMA, Thermo Fisher Scientific) for 48 h [32 (link)]. After differentiation, the cells were plated at a density of 2 × 10⁵ cells/well in 12-well plates and cultured in RPMI1640 medium supplemented with 10% (vol/vol) pooled healthy control plasma or pooled KD plasma from 10 individuals for 72 h; then, the cells were collected, and the RNA were extracted. We used three independent experiments for this study (N = 30 of KD patients and N = 30 of healthy controls). Real-time PCR was performed to quantify the mRNA levels of CASP1, CASP4, and CASP5.

After surgical resection, fresh tissue samples were immediately transferred to pre-cooled MACS Tissue Storage Solution (Miltenyi Biotec) and were shipped at 4 °C. For suspension mass cytometry, the tissue was processed as previously described^{29 (link)}. In brief, the Tumor Dissociation Kit, human and the gentleMACS Dissociator (both Miltenyi Biotech) were used for tissue dissociation, followed by filtering of the single-cell suspension. Cells were stained for viability with 25 mM cisplatin (Enzo Life Sciences) and subsequently fixed with 1.6% paraformaldehyde (Electron Microscopy Sciences) before storage at −80 °C. For scRNA-seq, an aliquot of the single-cell suspension was taken prior to viability staining and fixation and viable cells were frozen in RPMI-1640 cell culture medium (Thermo Fisher) supplemented with 10% fetal bovine serum and 10% DMSO (Sigma-Aldrich) using a Mr. Frosty Freezing Container (Thermo Fisher). Viable cell aliquots were stored in liquid nitrogen.

In a pilot experiment, 5 g of yard soil were spiked with 400 µL of infectious blood at a titer of 7.25 log₁₀ HAD₅₀/mL and stored at 4 °C or 25 °C. For comparison, a blood-only control was kept at the same conditions. Blood and soil were tested at time points 0, 3, 6, 24, 48, and 72 h, as well as 1, 2, 3 and 4 weeks. At the respective time points, 5 mL of RPMI-1640 cell culture medium (Thermo Fisher Scientific, Schwerte, Germany) with 10% fetal bovine serum (FBS) and 2% antibiotics (Gibco Penicillin–Streptomycin mix, 10,000 U/mL; Thermo Fisher Scientific) was added to the inoculated soil. Then the soil was agitated in the media by vortexing for 45 s (see Figure 6 for all steps). Next, soil and media were sonicated for 45 s at 4 °C with the settings duty cycle 40%, output 3.5 in a Branson Sonifier 450 (Heinemann Ultraschall- und Labortechnik, Schwäbisch Gmünd, Germany). After sonication, the soil suspension was centrifuged for 30 min at 2500× g at 4 °C. The supernatant was poured over a coffee filter, pushed through a 0.45 µm syringe filter (Millex Filter Units; Merck Millipore Ltd., Tullagreen, Ireland) and the filtrate was stored at −80 °C prior to real-time PCR, virus isolation and titration (see Section 4.7).

Two clinical M3 isolates of S. pyogenes, obtained during the collection of the Canadian patient material described above, were grown over night (16–18 h) in Todd-Hewitt broth complemented with 1.5% yeast extract. A clinical isolate of Escherichia coli, obtained from a sepsis patient, was also included in the study and was grown over night in Luria Broth medium. Before infection the bacterial cultures were centrifuged at 2500 rpm for 10 min and resuspended in RPMI 1640 cell culture medium supplemented with 5% heat inactivated FCS, 10 mM L-glutamine, and 25 mmol/L HEPES (all from Thermo Scientific, Hyclone).