Rpmi 1640 culture medium

Monocytes were isolated from the blood of healthy adult human donors using the Bøuym method as described in [72 (link)]. Blood samples were processed within two hours. The viability of isolated monocytes was measured by the cell morphology and flow cytometric annexin V-FITC and PI-A apoptosis detection kit II (BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer’s protocol. Monocytes were suspended in the RPMI-1640 culture medium that had been supplemented with the 1% antibiotic PSN (penicillin—streptomycin—neomycin) solution (GE Healthcare, Little Chalfont, UK) and used for further research. The study was approved by the local Bioethics Committee at the Centre of Postgraduate Medical Education (no. 26/PB/2018).

Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute (RPMI)-1640 culture medium and penicillin–streptomycin were purchased from GE Healthcare Life Sciences (Marlborough, MA, USA). IL-1β ELISA kit (Human 88-7261-22) and TRIzol reagent (15596018) was purchased from Invitrogen (Carlsbad, CA, USA). Lipopolysaccharide (LPS; L6529), palmitate (P9767) and MCC950 (PZ0280) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Pioglitazone was purchased from Takeda Pharmaceutical (AD-4833, Kanagawa, Japan). Ezetimibe was obtained from Cayman Chemical (16331, Ann Arbor, MI, USA). The triglyceride assay kit was purchased from Bioassay systems (ETGA-200, Hayward, CA, USA). Anti-F4/80 antibody was obtained from Abcam (ab6640, Cambridge, MA, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Georgiachem (MT 1036, Norcross, GA, USA).

Cells of lymph nodes draining the site of immunization (PLN) were obtained from rats immunized with 100 µl of emulsion made of MBP and CFA, as described above. PLN cells (PLNC) were obtained by mechanical disruption. The isolation took place at 4, 7, and 14 days post immunization (d.p.i.). Mesenteric lymph nodes (MLNs) and Peyer’s patches (PP) were isolated from non-immunized rats and from rats on 4, 7, and 14 d.p.i. Four MLN were isolated from each rat and MLN cells (MLNC) were prepared by mechanical disruption. PP were obtained from the small intestine and PP cells (PPC) were obtained by mechanical disruption. PLNC were cultured in RPMI 1640 culture medium (PAA Laboratories, Pasching, Austria) that was supplemented with 2% rat serum. The cells were seeded at 5 × 10⁶/ml/well in 24-well plates (Sarstedt, Nümbrecht, Germany) and stimulated with MBP (10 µg/ml). MLNC and PPC were grown in RPMI1640 medium supplemented with 5% fetal calf serum (PAA Laboratories). MLNC (2.5 × 10⁶/ml) and PPC (2 × 10⁶/ml) were stimulated with concanavalin A (ConA, Sigma-Aldrich, 2.5 µg/ml). Cultures lasted for 24 h and subsequently cell culture supernatants were collected and kept frozen until assayed. Supernatants were also obtained from SCHs (1 g of spinal cord homogenized in 2 ml of PBS) after centrifugation on 10,000 g for 20 min.

Human polymorphonuclear cells (PMNs) were freshly isolated from Na-citrate-treated blood of healthy donors. For neutrophil-isolation, dextran-sedimentation and density gradient centrifugation using Ficoll-Paque Plus (Amersham Bioscience) was used according to the manufacturer's instruction. Cell purity was determined by Giemsa staining and was always above 99%. PMNs were suspended to a final density of 1×10⁶ cells/0.5 ml in RPMI 1640 culture medium (PAA Laboratories GmbH) supplemented with 10% heat-inactivated FCS (PAA Laboratories GmbH) and immediately used for the experiments. All incubations were performed at 37°C in humidified air with 5% CO₂.
To measure cell death induction 1x10⁶ cells/0.5 ml PMNs were cultured at 37°C in a 5% CO₂ atmosphere in 24-well plates and bacterial supernatants were added as indicated. After 1 h cell death induction was analyzed as described previously [78 (link),79 (link)].

The PCC test was performed as previously described [17 (link)]. To summarize, whole blood (0.5 ml) was added to 4.5 ml of RPMI 1640 culture medium (PAA Laboratories GmbH, Pasching, Austria) and stimulated by adding PHA (10 mg/mL) (Sigma-Aldrich, St. Louis, MO, United States) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Carlsbad, CA, USA) and antibiotics—100 U/ml penicillin and 100 g/ml streptomycin (Polfa Tarchomin, Warsaw, Poland). The cultures were incubated for 48 h at 37 °C and 5% CO₂. Exactly 30 minutes before ending the culturing process, calyculin A (50 nM) was added to the culture medium causing the induction of PCC in any PHAse of the cell cycle (G1, S, G2, M, and A). After 48 hours of cell culture, the cells were harvested according to previously published procedures [17 (link)]. The samples were dropped onto an ethanol-washed microscopic slide. Fixed samples were stained with 4% Giemsa in phosPHAte-buffered water and stored at room temperature until examination. Each measurement point was prepared in triplicate. All slides were coded and blinded to the scorer. Decoding was completed only after the microscopic examination of all slides from the study.

Whole blood was obtained in EDTA containing tubes under sterile conditions. 300 μL whole blood were added to 300 μL RPMI 1640 culture medium (PAA Laboratories, Pasching, Austria) supplemented with 2 mM L-glutamine, 50 μg/mL penicillin/streptomycin, 5 mM HEPES, 10 μM mercapto-ethanol and 0.1% fetal calf serum, and seeded in 24-well plates (Greiner, Nürtingen, Germany) at 37°C (5% CO₂/95% air atmosphere). For stimulation, 1 ng/mL lipopolysaccharide (LPS, Sigma-Aldrich, Taufkirchen, Germany) was added to culture medium. Supernatants were collected after 24 h and stored at -20°C until analyses. LPS was chosen for stimulation because of its important role for immunological recognition of and response to uropathogens, i.e. E. coli [2 (link)]. Levels of pro-inflammatory interleukin-1ß (IL-1β), IL-8, in the culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA) according to manufacturer‘s protocol and the alarmin S100A8/S100A9 was measured by a home-made ELISA as described in [15 (link)].

Human peripheral blood mononuclear cells (PBMCs) were isolated from EDTA-blood of healthy donors by the Ficoll/Isopaque (Pharmacia, Uppsala, Sweden) density gradient centrifugation. Monocytes were separated from PBMCs by counter-flow centrifugal elutriation with the JE-6B elutriation system equipped with a 5 ml Sanderson separation chamber (Beckman-Coulter, Palo Alto, CA), as previously described [19 (link)]. Monocytes were suspended in RPMI 1640 culture medium (PAA Laboratories, Pasching, Germany) with gentamycin (Sigma, St. Louis, MO) (25 μg/ml). Purity of monocytes was over 95 %, as judged by staining with anti-CD14 mAb (BD Biosciences Pharmingen, San Diego, CA) and flow cytometry analysis (FACSCanto BD Biosciences Immunocytometry Systems, San Jose, CA). The study was approved by the local Jagiellonian University Ethical Committee (No. KBET/160/b/2011).