Hepes solution

Fetal mouse lung organoids were derived as previously reported from bud tips with several modifications [14 (link)]. Briefly, mesenchyme-free epithelium rudiments containing at least 3 bud tips were embedded in 20 μL MRF in a 24-well and incubated with advanced DMEM/F12 supplemented with 1% p/s, 1 mM HEPES solution, 1% Glutamax, 1% ITS (all from Thermo Fisher Scientific), Heparin solution (Sigma-Aldrich), 50 ng/mL mouse recombinant Fgf10, 50 ng/mL mouse recombinant Fgf9, 50 ng/mL mouse recombinant Egf (Peprotech), 3 μM CHIR99021, 1 μM p38-MAPK pathway inhibitor BIRB-796, 1 μM A83-01 (all from Tocris), 10 μM Y-27632 Rock-Inhibitor (Cambridge Bioscience). Organoids were expanded by mechanical dissociation and cryopreserved in 50% advanced DMEM/F12, 40% FBS, and 10% DMSO.

Human colon adenocarcinoma cell lines SW480 and COLO205 were obtained from ATCC Cell Line Collection and THP 1-ASC-GFP monocytes were obtained from Invovogen. The SW480 and COLO205 cell lines were maintained in RPMI 1640 (ThermoFisher Scientific) supplemented with 10% FBS, 1% L-Glutamine (ThermoFisher Scientific), 10 mM HEPES solution (ThermoFisher Scientific) and 1 mM sodium pyruvate (ThermoFisher Scientific). THP 1-ASC-GFP cells were cultured in RPMI-1640 with L-Glutamine (Merck), 10% heat inactivated premium grade FBS (Biowest), 10 mM HEPES, 1 mM sodium pyruvate (Merck), 0,45% glucose (Merck) and 100U/ml penicillin–streptomycin (Merck) at 37 °C and 5% CO₂. Zeocin (200 µg/ml) (Invivogen) was added to the culture medium as per the manufacturer’s instructions. Cell density was maintained between 5 × 10⁵ and 1.5 × 10⁶ cells/ml and cells were used up to passage number ten. Differentiation was conducted with 100 ng/ml PMA (Merck) for 72 h followed by 72 h of rest in fresh media. Priming of undifferentiated or differentiated cells was conducted with 500 ng/ml ultrapure LPS (Invivogen) for up to 24 h as indicated. Activation of inflammasome complex formation was conducted with 5 mM ATP (Merck) for 30 min.

For FRAP, cells were plated onto glass-bottomed dishes (35 mm glass-bottomed μ-dishes, IBIDI) and transfected as described above. 36 to 48 h later, 1M HEPES solution (ThermoFisher Scientific) was added to the medium to a final concentration of 20 mM, to buffer the medium in air. Cells were imaged on an LSM 880 Airyscan inverted confocal microscope (Zeiss), using the 40x objective lens (NA 1.4) in Airyscan mode. A stage-top incubator maintained the cells at 37 °C. A small circular region (∼1 μm in diameter) was bleached using a 488 nm wavelength laser set to 100% for ∼50 iterations to ensure levels of fluorescence in this region were reduced to very low levels. Fluorescence recovery was then allowed to proceed for approximately 2 min, until recovery had plateaued, at a frame rate of one per second. Data was analyzed using the FRAP plugin in the Zeiss software. Fluorescence recovery curves were fit with a single exponential to obtain values of t_1/2 and immobile fraction. For each mutation, at least 10 cells over three separate experiments, were analyzed. Results from the replicates were combined, graphs generated, and statistical analysis (ANOVA) was performed using GraphPad Prism.

Cytarabine (ara‐C) was purchased from Jena Bioscience, Germany (cat no. N‐20307‐5), and Sigma‐Aldrich, Sweden (cat no. C1768), gemcitabine (dF‐dC; cat no. G6423), hydroxyurea (HU; cat no. H8627), triapine (3‐AP; cat no. SML0568), clofarabine (Cl‐F‐ara‐A; cat no. C7495), fludarabine (2‐F‐ara‐A; cat no. F2773) and cladribine (2CdA; cat no. C4438), were purchased from Sigma‐Aldrich, Sweden, and MK‐1775 (cat no. SC‐483196) was purchased from Santa Cruz Biotechnology, USA. Compounds were typically prepared as 10–50 mM stock solutions in DMSO and were stored at −20°C, with the exception of HU which was prepared fresh. Alternatively, when not being used with liquid handling equipment, ara‐C and HU were prepared in water; no difference in EC₅₀ was observed between DMSO and water stocks. N‐acetyl cysteine (NAC; Sigma‐Aldrich) was dissolved in 1 M HEPES solution (Thermo Fisher Scientific) to a concentration of 0.5 M and pH adjusted to 7.2. Prior to use, this stock solution was diluted to 5 mM in complete cell media.

DMEM, MEM, Neurobasal media, N2 supplement, penicillin/streptomycin, trypsin-EDTA, DNAse, HEPES solution, sodium bicarbonate, non-essential amino acid solution, GFAP antibody, and Prolong Anti-fade with DAPI were all purchased from Thermo Fisher Scientific (Waltham, MA, USA). Fetal bovine serum was purchased from Atlanta biologicals (Lawrenceville, GA, USA). Zika NS1 rabbit polyclonal antibody (GTX133306) was purchased from Genetex (San Antonio, TX, USA). Zika Virus strain PA259459 was obtained from the World Reference Center for Emerging Viruses and Arboviruses through the University of Texas Medical Branch (UTMB) at Galveston (Galveston, TX, USA). All other materials were purchased from Sigma-Aldrich (St. Louis, MO, USA).

All cell lines were cultured in complete RPMI medium (Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO), 1 mM sodium pyruvate (Thermo Fisher Scientific), 50 μM 2-mercaptoethanol (Nacalai Tesque), 1% GlutaMAX solution (Thermo Fisher Scientific), 1% MEM non-essential amino acids (Thermo Fisher Scientific), 10 mM HEPES solution (Thermo Fisher Scientific), 50 μM 2-mercaptoethanol (Nacalai Tesque), 100 units/ml penicillin, and 100 μg/ml streptomycin (Nacalai Tesque, Kyoto, Japan). Mycoplasma contamination is regularly checked using PlasmoTest mycoplasma detection kit (InvivoGen, San Diego, CA).

PBMCs were isolated from Trima Cones (Innovative Blood Resources, Saint Paul, MN, USA) using a Ficoll-Paque gradient. Briefly, Trima Cones were washed with phosphate-buffered saline (PBS) (B30250, Bio-Techne, Minneapolis, MN, USA) to remove the plasma layer, then carefully layered on top of the Ficoll-Paque PLUS (17544202, Cytiva, Marlborough, MA, USA) and centrifuged for 30 min at 800×g. The PBMC layer was transferred to a separate tube and washed with PBS, before a 10-min incubation with red blood cell lysis buffer (FC002, Bio-Techne). The lysis was quenched with 0.5 M HEPES solution (15630-80, ThermoFisher, Waltham, MA, USA) and washed twice with PBS. The PBMC population was raised in Cryostor CS10 (210102, BioLife Solutions, Bothell, WA, USA) then aliquoted for freezing. The aliquots were arranged in a cryo-freezing container (5100-001, Nalgene, Rochester, NY, USA) which was placed in a −20°C freezer for 30 min, then transferred to a −80°C freezer overnight. The following morning, the frozen aliquots were transferred to a liquid nitrogen storage tank (CMR-2800, ThermoFisher) until use.