Fbs superior

Isomeric purity of (Z)-4-undecenal (Z4-11Al) was 98.6%, according to gas chromatography coupled to mass spectrometry (6890 GC and 5975 MS, Agilent Technologies, Santa Clara, CA, USA). Isomeric purity of (Z)-4-nonenal (Z4-9Al) and (Z)-6-undecenal (Z6-11Al) were 97.4 and 96%, repectively. Chemical purity of these synthetic aldehydes was >99.9%. Ethanol (redistilled; Merck, Darmstadt, Germany) was used as solvent.
For the OR screening assays, the following chemicals were used: Dulbecco’s MEM medium (#F0435), FBS superior (#S0615), L-glutamine (#K0282), penicillin (10000 U/ml)/streptomycin (10000 μg/mL) (#A2212), trypsin/EDTA solution (#L2143) (Biochrom, Berlin, Germany), CaCl2∗2H2O (#22322.295), D-glucose (#101174Y), dimethyl sulfoxide (DMSO) (#83673.230), HEPES (#441476L), potassium chloride (#26764.230), and sodium hydroxide (#28244.295) (VWR Chemicals BDH Prolabo, Leuven, Belgium), sodium chloride (#1064041000, Merck, Darmstadt, Germany), ViaFect™ Transfection Reagent (#E4981, Promega, Walldorf, Germany), D-luciferin (beetle) monosodium salt (#E464X, Promega, Walldorf, Germany), Pluronic® PE 10500 (#500053867, BASF, Ludwigshafen, Germany), (R)-(−)-carvone (#W224908, Sigma-Aldrich, Steinheim, Germany).

The monocytic cell line THP-1 (DSMZ No. ACC 16) was cultured in RPMI 1640 (Biochrom AG, Berlin, Germany) supplemented with 10% FBS superior (Biochrom AG), 2 mM l-glutamine (Biochrom AG), 1 mM Na-pyruvate (Biochrom AG), gentamycin (10 µg/mL) (Biochrom AG), and 1 mM HEPES buffer (Biochrom AG) at 37 °C in a 5% CO₂ humidified atmosphere and passaged 2–3 times per week. Cells were used up to passage 20. To perform infection experiments, cells were differentiated into macrophages by stimulation with phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich, Munich, Germany) 48 h prior to infection. For that purpose, cells were seeded in a 10 µM PMA solution in RPMI including listed supplements but without antibiotics at a density of 1 × 10⁶ cells per well, applying a 1.5-mL volume together with a six-well cell culture plate (Sarstedt AG, Nümbrecht, Germany). After 24 h of PMA stimulation, the stimulus was removed by washing the adherent cell layer with PBS. Medium including listed supplements but without antibiotics was provided for another 24 h before using THP-1-derived macrophages for infection experiments.

Human immortalized C28/I2 cells, originating from cells isolated from rib cartilage,21 were cultured in Dulbecco's modified Eagle's medium (DMEM)/HAM's F‐12 medium (Biochrom, Darmstadt, Germany) supplemented with 5 % fetal bovine serum (FBS Superior; Biochrom) and antibiotic‐antimycotic solution (100 U/ml penicillin, 0.1 mg/ml streptomycin, 0.25 µg/ml amphotericin‐B; Sigma‐Aldrich) in a humidified atmosphere at 37 °C and 5 % CO₂.
Human primary chondrocytes were isolated from total knee arthroplasty samples with informed consent and ethical approval by the Ethics Committee of Salzburg (415‐E/1965/4‐2015). All patients were female and all samples were macroscopically graded between 3 and 4 or 4 (Outerbridge classification). Cartilage samples of the whole tibial area and femur condyles were removed from subchondral bone, minced, washed in phosphate‐buffered saline (PBS) and digested overnight in DMEM/HAM's F‐12 supplemented with FBS and 2 mg/ml collagenase Type II (275 units/mg; Thermo Fisher Scientific) at 37 °C on a shaker. After 24 h, the cells were centrifuged, washed twice with PBS, resuspended in medium and seeded as required for the following experiments. For 7‐hydroxy‐3H‐phenoxazin‐3‐one‐10‐oxide sodium salt (Resazurin) assays and Fura‐2 assays, cells were always used at the first passage.

Human immortalized C28/I2 cells (Goldring et al., 1994 (link); Finger et al., 2003 (link)) were cultured in 25 cm² flasks with DMEM/HAM’s F-12 medium (Biochrom, Berlin, Germany) supplemented with 5% fetal bovine serum (FBS Superior, Biochrom) and antibiotic-antimycotic solution (100 U/ml penicillin, 0.1 mg/ml streptomycin, 0.25 μg/ml amphotericin-B; Sigma–Aldrich). C28/I2 cells were kept at 37°C in a humified atmosphere of 5% CO₂ (standard culture conditions). Subcultures were established once a week until passage 25. Primary chondrocytes were isolated from total human knee arthroplasty samples with informed consent and ethical approval by the Ethics Committee of Salzburg (415-E/1965/4-2015) as described in Winklmayr et al. (2019) (link). In brief, cartilage samples were removed from subchondral bone and crashed into small pieces. After washing with phosphate-buffered saline (PBS), they were put on a shaker and digested overnight in DMEM/HAM’s F-12 supplemented with FBS and 2 mg/ml collagenase (Thermo Fisher) at 37°C. After 24 h, cells were (1) centrifuged, (2) washed twice with PBS, (3) resuspended in medium, and (4) seeded as required for the following experiments. Primary chondrocytes were used up to passage 2.

Murine ES cell lines described previously [57 (link)] were grown in high glucose DMEM with stable glutamine (GIBCO) containing 10 % FBS Superior (Biochrom), 100 μM non-essential amino acids (GIBCO), 1 % Penicillin/Streptamycin (GIBCO) and 100 μM β-Mercaptoethanol (Sigma) in presence of 1000 U/mL of Leukemia inhibitory factor (LIF, Milllipore). Differentiation of aCaBs was performed in hanging drop culture for two days using 1000 cells as starting material for one EB in Iscove’s basal medium (Biochrom) containing 10 % FBS (Biochrom), 100 μM non-essential amino acids (GIBCO), 1 % Penicillin/Streptamycin (GIBCO) and 450 μM 1-Thioglycerol. For additional 4 days, the cells were differentiated in suspension culture, and at day 6 of differentiation consistently 15 EBs were seeded on one well of a 24-well-plate. Antibiotic selection with 400 μg/mL G418 (Biochrom) was initiated at day 8 post seeding. 4 days thereafter, aCaBs were isolated via treatment with 6000 U/mL Collagenase IV (GIBCO) for 30 min. To obtain single cells for subsequent experiments, the bodies were further dissociated with 100 % Accutase (Affimetrix) for 15 min. To ensure successful generation of aCaBs, potential mycoplasma contamination was routinely controlled twice a week using the PCR based MycoSPY kit system (Biontex).

The following chemicals were used: Dulbecco’s MEM medium (#F0435), FBS superior (#S0615), L-glutamine (#K0282), penicillin (100U/ml)/streptomycin (100U/ml) (#A2212), trypsin/EDTA solution (#L2143) (Biochrom, Berlin, Germany), MEM non-essential amino acid solution (100x) (#M7145, Sigma-Aldrich, Steinheim, Germany), Gibco^® HAT supplement (#21060-017, Thermo Fisher, Dreieich, Germany),CaCl₂*2H₂O (#22322.295), D-glucose (#101174Y), dimethyl sulfoxide (DMSO) (#83673.230), HEPES (#441476L), potassium chloride (#26764.230), and sodium hydroxide (#28244.295) (VWR Chemicals BDH Prolabo, Leuven, Belgium), sodium chloride (#1064041000, Merck, Darmstadt, Germany), D-luciferin (beetle) monosodium salt (#E464X), HaloTag^® Alexa Fluor^® 488 Ligand (#G1001, Promega, Madison, USA), Dynasore (#2897, Tocris Bioscience, Bristol, UK), Hoechst33342 (#1399, Invitrogen, Eugene, USA), Mowiol 4-88 (#0713, Carl Roth GmbH + Co. KG, Karlsruhe, Germany), Paraformaldehyde (PFA) (#18814, Polysciences Inc., Warrington, USA).
Odorants were purchased from Sigma-Aldrich (Steinheim, Germany), Alfa-Aesar (Karlsruhe, Germany) and Chemos GmbH (Regenstauf, Germany) (Table S1).