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89 protocols using DNase I

Inguinal lymph nodes were mechanically disaggregated and digested in 1 mL non-supplemented RPMI 1640 medium (ThermoFisher Scientific, ref 61870-036) containing 5 mg/mL of collagenase type IV (Gibco, ref 17104019) and 5 µg/mL of DNAse I (AppliChem, ref A3778,0010) for 30 min at 37 °C. Single-cell suspension was obtained using a 70 μm cell strainer (BD Falcon, ref 352350). For skin preparations, vaccinated skin was excised, cut in small fragments and digested in 1 mL non-supplemented RPMI 1640 medium (ThermoFisher Scientific, ref 61870-036) containing 5 mg/mL of collagenase type IV (Gibco, ref 17104019) and 5 µg/mL of DNAse I (AppliChem, ref A3778,0010) for 30 min at 37 °C, skin pieces were then disaggregated mechanically using microscope slides with ground edges (Sail Brand, ref 7105) and single-cell suspension was obtained using a 70 μm cell strainer (BD Falcon, ref 352350) followed by a second digestion with 1 mL of supplemented of RPMI 1640 medium containing 5 µg/mL of DNAse I (AppliChem, ref A3778,0010) during 5 min on ice.
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Dissected liver tissues were incubated for 30 min at 37°C in dissociation medium composed of Leibovitz-15 medium (Sigma-Aldrich), 45% glucose, 1 g/L DNAse I (Applichem) and collagenase A (Roche, Basel, Switzerland). Digested liver fragments were passed through a nylon cell strainer, and cells free in suspension were collected in 50 ml wash medium/liver and stored at 4°C. Cells were centrifuged at 30 × g for 15 min at 4°C to pellet parenchymal cells, i.e., hepatocytes. Parenchymal cells were washed 3 times with FACS buffer and analyzed by flow cytometry.
Non-parenchymal cells (NPCs) remaining in suspension were collected and centrifuged at 300 × g for 10 min at 4°C. Pelleted cells were resuspended in ice-cold-HBSS, mixed with freshly prepared 30% Histodenz (Sigma-Aldrich), and centrifuged at 1,500 × g for 20 min at 4°C. The NPCs at the Histodenz interface were collected, washed, and suspended in FACS buffer for analysis. The NPCs were phenotyped using dye-conjugated monoclonal antibodies specific for CD45 (clone 30-F11), F4/80 (clone BM8), and CD31 (clone 390) (BD Biosciences, Franklin Lake, NJ, United States). Stained samples were acquired on a multichannel cytometer BD LSR II equipped with FACS Diva software (BD Biosciences; Franklin Lake, NJ, United States) and analyzed using FlowJo 7.6.5 (Becton, Dickinson Company; Ashland, OR, United States).
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Male mice germ cells were purified following a well-establish protocol40 (link),53 (link),54 (link). In brief, mice were sacrificed, and the testes were decapsulated. The seminiferous tubules were isolated from other testicular tissues by incubation with 0.2% W/V collagenase type I (Cat. 17100017, Gibco, MA, USA) and 1 mg/mL DNase I (Cat. A3778, Applichem, IOWA, USA) in a water bath at 37 °C for 10 min, after which the samples were centrifuged at 1200 rpm for 2 min. The pellet containing the seminiferous tubules was resuspended in 5 ml of 0.25% trypsin (Cat.15050057, Gibco, MA, USA) containing 1 mg/mL DNase I at 37 °C for 5 min with gentle shaking. The sample was centrifuged at 1500 rpm for 5 min, after which the pelleted cells were resuspended in 20 mL of high glucose DMEM (Cat. 12100046, Gibco, MA, USA) containing 0.5% BSA and filtered through a 40 μm nylon cell strainer (Cat. 352340, Falcon®, BD, NJ, USA). The recovered cells in the filtrate were resuspended in 20 mL DMEM containing 0.5% BSA and loaded into a cell separation apparatus (BOMEX Corporate) containing a 2–4% BSA gradient in 600 mL of DMEM. After sedimentation for 3 h, the cells were collected into tubes from the bottom of the separation apparatus at a rate of 10 mL/min. The cell type and purity in each fraction were assessed according to their diameter and morphological characteristics under a light microscope.
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4

Extraction and Characterization of Chondroitin and Hyaluronic Acid

In order to extract intracellular chondroitin and HA, about 10 g of wet cells were resuspended in 20 mL of distilled water and autoclaved at 120 °C for 15 min. After centrifugation the supernatant was recovered and precipitated on ice with 4 volumes of cold ethanol 96% v/v, and stored at 4 °C o/n. The pellet, recovered after centrifugation was treated with 1 mg/mL DNase I (Applichem) in a buffer containing 100 mM Tris pH 7.5, 50 mM MgCl2 and 10 mM CaCl2 for 1 h at 37 °C and successively digested with 2.5 mg/mL of Protease K from Aspergillus (Sigma-Aldrich) for 2 h at 56 °C. A second precipitation was repeated on the sample o/n at 4 °C and the resulting pellet was dried and used for: (a) hydrodynamic characterization (b) quantification of uronic acids through carbazole assay (Bitter and Muir 1962 (link)) (c) determination of relative ratios of HA and chondroitin through high performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) monosaccharide determinations after hydrolysis.
The powder obtained from the control sample was further purified to verify the production of chondroitin in S. equi subsp. zooepidemicus-pNZ8148kfoAkfoC, through structure determination by NMR analysis.
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5

Isolation and Stimulation of Fibroblastic Stromal Cells

PPs or mLNs were dissected into small pieces and transferred into a 24-well dish filled with RPMI 1640 medium containing 2% FCS, 20 mM HEPES (all from Lonza), 1 mg/ml collagenase D (Sigma) and 25 μg/ml DNaseI (Applichem). Dissociated PPs or LNs were incubated at 37 °C for 30 min. After enzymatic digestion, cell suspensions were washed with PBS containing 0.5% FCS and 10 mM EDTA. To enrich for fibroblastic stromal cells, hematopoietic cells were depleted by incubating the cell suspension with MACS anti-CD45 and anti-Ter119 microbeads and passing over a MACS LS column (Miltenyi Biotec). Cell sorting was performed using a FACSAria cell sorter (BD Biosciences) or a S3 cell sorter (Biorad). For in vitro assays, PP or mLN fibroblastic stromal cells were cultured for 7 d in RPMI with 10% FCS, and 5 × 104 cells were stimulated with 100 ng of R848 or left untreated. Supernatants were collected after 24 h, and IL-6 and CCL2 concentrations were determined by cytomeric bead array (CBA, BD biosciences) and IL-15 concentration was determined by ELISA (Abcam).
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The following reagents were used in the experiments described below: docosahexaenoic acid (DHA), linoleic Acid (LA), 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) and resolvin D1 (RvD1) (Cayman chemicals, Ann Arbor, Michigan). Additional materials used included Collagenase II (Worthington Biochemical, Lakewood, NJ), DNase I (AppliChem, Ottoweg Darmstadt), RPMI 1640, and DMEM/F12 media fetal bovine serum (FBS) (Thermo Fischer Scientific, Grand Island, NY) Bovine serum albumin (BSA; Fraction V; Sigma Aldrich St Louis, MO) and radio-immunoprecipitation assay (RIPA) lysis buffer (Sigma Aldrich St Louis, MO). The protease inhibitor cocktail was purchased from Roche GmbH, Germany.
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Mouse tissue‐derived cells were isolated from cell suspension after digestion of minced tissue with 1 mg/ml collagenase IV (GIBCO), 0.5 mg/ml Dispase (GIBCO), and 1 mg/ml DNase I (Applichem). Mouse blood cells were derived from peripheral blood after red blood cell lysis. Human immune cells were derived from density gradient separated blood polymorphonuclear cell and mononuclear cell fractions using Lympholyte‐poly solution (Cedarlane), and only autologous, sample‐matched cells were used for co‐culture. Specific cell populations were enriched by either magnetic or fluorescence‐based (FACS) sorting for cell type‐specific antibodies (see Appendix Tables S1 and S3 for detailed information). Biotinylated antibodies with MACS anti‐biotin magnetic beads and MACS columns (Miltenyi Biotec) were used for magnetic sorting. FACS sorting was done on FACS‐Aria II or FACS‐ARIA Fusion and FACS analysis on LSR Fortessa machines (all BD Biosciences).
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MLNs from Cxcl13-Cre/TdTomato mice were collected and mechanically dissociated in a 24-well dish filled with RPMI 1640 medium containing 2% FCS, 20 mM HEPES (all from Lonza), 1 mg ml−1 collagenase P (Sigma-Aldrich), 25 µg ml−1 DNase I (Applichem) and dispase (Roche). Dissociated tissues were incubated at 37 °C for 30 min. After enzymatic digestion, cell suspensions were washed with PBS containing 0.5% FCS and 10 mM EDTA. For the in vitro assays, LN fibroblasts were cultured for 10 d in RPMI 1640 supplemented with 10% FCS, 1% penicillin-streptomycin and 16 µg ml−1 gentamicin. To evaluate the differentiation and activation potential, 1.5 × 104 cells were plated in 24-well plates and stimulated for 48 h with recombinant mouse TGFβ1 (10 ng ml−1, catalog no. 7666-MB/CF, R&D Systems), recombinant mouse PRGN (10 ng ml−1, catalog no. 2557-PG, R&D Systems), recombinant mouse VEGF (25 ng ml−1, catalog no. 493-MV/CF, R&D Systems), recombinant mouse IL-4 (10 ng ml−1, catalog no. 404-ML/CF, R&D Systems) or recombinant mouse IL-1β (1 ng ml−1, catalog no. ab259421, Abcam). Supernatants were collected to determine the IL-6 concentration using the Mouse Inflammation Cytometric Bead Array Kit (BD Biosciences) according to the manufacturer’s instructions. Cells were collected and the expression of TdTomato on EYFP+PDPN+ cells was determined by flow cytometry.
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We used the magnetic bead method described by Takemoto et al. 2006 with appropriate modifications [23 (link)]. Briefly, kidneys were dissected together with the abdominal aorta and transferred into dishes filled with 37°C pre-warmed Hank’s buffered salt solution (HBSS). Each kidney was perfused slowly through the renal artery with 4 ml 37°C warm bead solution and 1 ml bead solution plus enzymatic digestion buffer [containing: collagenase 300 U/ml (Collagenase Type II, Worthington, Lakewood, New Jersey, USA), 1 mg/ml pronase E (P6911, Sigma, Schnelldorf, Germany) DNase I 50 U/ml (A3778, Applichem, Darmstadt, Germany)]. Kidneys were minced into 1 mm³ pieces using a scalpel. After addition of 3 ml digestion buffer they were incubated at 37°C for 15 min on a rotator (100rpm). The solution was pipetted up and down with a cut 1000μl pipette tip every 5 min. After incubation all steps were performed at 4°C or on ice. The digested kidneys were gently pressed twice through a 100 μm cell-strainer and the flow through was washed extensively with HBSS. After spinning down, the supernatant was discarded and the pellet resuspended in 2 ml HBSS. These tubes were inserted into a magnetic particle concentrator and the separated glomeruli were washed twice. Podocytes were isolated as previously described [24 (link)].
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GFP-H3 wt and R2A mutant constructs were co-expressed in HEK 293T cells with UHRF1-His and harvested after treatment with 2 mM N-ethylmaleimide (NEM, AppliChem) for 5 min. Lysates were prepared by firstly isolating nuclei in hypotonic buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM MgCl2, 10% glycerol, 0.1 mM EDTA, 0.1 mM DTT, 1× protease inhibitor, 2 mM PMSF, 0.1% NP-40, 0.625 mg/ml NEM) and secondly by lysis of the nuclei in hypertonic buffer (20 mM HEPES pH 7.9, 150 mM KCl, 1.5 mM MgCl2, 10% glycerol, 0.1 mM EDTA, 1 mM DTT, 1× protease inhibitor, 2 mM PMSF, 1 mg/ml DnaseI (AppliChem), 0.625 mg/ml NEM). Prior to immunoprecipitation, the GFP concentration was equalized using lysates from UHRF1-His transfected HEK 293T cells for dilution. After immunoprecipitation of GFP-H3 with the GFP-Trap (Chromotek) and washing (20 mM HEPES pH 7.9, 300 mM KCl, 10% glycerol, 0.1% Triton X-100), the bound fraction was analyzed by western blot.
For semiquantitative analysis of the GFP-H3 wt or K18A, K23A, K18A-K23A and R2A ubiquitination, the GFP fusion constructs were co-expressed with HA-ubiquitin in HEK 293T cells and 2 days after transfection, the cells were harvested as described above and further processed as reported previously29 (link).
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