Samples were washed with calcium-free and magnesium-free phosphate-buffered saline and then separated into thin layers and cut into small pieces on ice. The tissues were then digested using an enzyme cocktail, which consisted of 3 mg/ml collagenase type I (Sigma–Aldrich), 0.156 mg/ml collagenase type XI (Sigma–Aldrich), 0.25 mg/ml soybean trypsin inhibitor (Worthington), 0.1875 mg/ml lyophilized elastase (Worthington), 0.24 mg/ml hyaluronidase type I (Sigma–Aldrich) and 60 U/ml DNase I (Sigma–Aldrich) (6 (link), 13 (link)). Tissues were digested in a 37°C water bath for 30 min, the supernatants were collected, with Dulbecco's modified Eagle's medium (DMEM) and 10% fetal bovine serum added to stop the reaction. Fresh enzyme cocktail was then added to the digested tissues for a second round of digestion. The digestion process was repeated 3–4 times. Then, the erythrocytes were removed via ACK lysis buffer (Lonza) treatment for 5 min. The CD45+ cells were then purified using a magnetic cell sorting system (Miltenyi Biotec) according to the manufacturer's instructions. Following centrifugation (300 g, 4°C, and 5 min), the cell pellet was collected. Finally, 10 μl of suspension was counted under an inverted microscope using a hemocytometer. Trypan blue was used to quantify live cells.
Hyaluronidase type 1
Manufactured by Merck Group
Sourced in Germany
Hyaluronidase type I is an enzyme that catalyzes the depolymerization of hyaluronic acid, a major component of the extracellular matrix. It is used as a laboratory tool to modify the viscosity of hyaluronic acid-containing samples.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase type 1, by Merck Group (4 mentions)
Dnase 1, by Roche (3 mentions)
Dnase 1, by Merck Group (2 mentions)
Collagenase type xi, by Merck Group (2 mentions)
Papain, by Merck Group (2 mentions)
Collagenase type 2, by Merck Group (2 mentions)
Collagenase type 1, by Fujifilm (2 mentions)
Magnetic cell sorting system, by Miltenyi Biotec (1 mentions)
Ack lysis buffer, by Lonza (1 mentions)
Soybean trypsin inhibitor, by Worthington (1 mentions)
Collagenase type 4, by Merck Group (1 mentions)
Dmem glutamax, by Thermo Fisher Scientific (1 mentions)
6 well plate, by Corning (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Collagenase a, by Roche (1 mentions)
Facscalibur, by BD (1 mentions)
Cd45 fitc, by BioLegend (1 mentions)
14 15 eet, by Cayman Chemical (1 mentions)
Pe anti mouse cd11b, by BioLegend (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
12 protocols using hyaluronidase type 1
1
Isolation of CD45+ Cells from Tissue
2
Xenograft Tumor Dissociation and Immune Cell Isolation
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Fresh xenograft tumours were soaked in PBS and chopped to 1 mm3 size with surgical blade. After washed with PBS, the slices were digested with collagenase (type IV, 1.5 mg/ml, Sigma) and hyaluronidase (type I, 0.25 mg/ml, Sigma) DMEM culture medium by rotating at 37 °C for 2 h. Digested cells were filtered through a sterilised cell strainer (70 µm) and collected by centrifugation at 800 rpm/min for 5 min. After washed in 25 ml PBS, the isolated cells were either cultured for ~48-h for the second round of xenograft growth or resuspended in 5 ml RBC lysis buffer for 10 min. After washed in PBS, the cells were then aliquoted in 106 cells/100 µl culture medium for flow cytometry analysis of F4/80+ and CD206+ expressions.
3
Isolation of Vascular Smooth Muscle Cells
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The in vitro analyses of VSMCs were performed with isolated VSMCs obtained from the aorta of global CRP4−/− and CRP4+/+ mice from both sexes at the age of 6–8 weeks. After euthanasia by CO2 inhalation, the aorta was isolated and cleaned from adventitial tissue. VSMC cultures were established from four aorta per genotype, which were pooled for the enzymatic dissociation as described previously [44 (link)]. In brief, aorta were cut into ~1 mm segments and digested by Papain (0.7 mg/mL from Sigma Aldrich) for 1 h at 37 °C following by Hyaluronidase Type I (1 mg/mL) with Collagenase Type II (1 mg/mL) (both from Sigma Aldrich) for 25 min at 37 °C. Digestion was stopped with VSMC culture media (10% FBS, 5% Penicillin in DMEM Glutamax® from Thermo Fisher Scientific), and the cells were centrifuged for 7 min at 300× g. Sedimented cells were resuspended in 1 mL VSMC culture media, counted using a Neubauer haemocytometer (Merck Millipore) and seeded in 6-well plates (Corning GmbH, Kaiserslautern, Germany) at a density of 5× 104 cells/mL. The medium was changed every third day until the experiments with primary VSMCs or highly passaged cells (P10-15) were performed. For analyses of primary and passaged VSMC, results were obtained from multiple (at least three) independent isolates of each genotype.
4
Cytokine Production in Mononuclear Cells
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Mononuclear cells (MNCs) were prepared from 4 groups of mice to determine cytokine production according to our previous reports [17 (link)–19 ]. Mediastinal lymph nodes (MLNs) tissues and spleen were removed from the mice. The removed tissues were chopped by sterile scissors, and digested in a 37°C water bath for 2 hours in digestion buffer containing 1.5 mg/mL collagenase A (type IA, Boehringer Mannheim, Mannheim, Germany), 0.02 mg/mL DNase I (type I, Boehringer Mannheim), and 0.75 mg/mL hyaluronidase (type I, Sigma). After the digestion procedure, the cell pellets were filtered using a metal mesh. Then, filtered digestives were washed 3 times by RPMI 1640 (Gibco BRL, Rockville, MD) containing 10% fetal bovine serum (FBS, Gibco BRL) and 1% penicillin/streptomycin (PC/SM, Gibco BRL), followed by the density gradient method to purify the MNCs. These MNCs were cultured at a density of 1×106/200 μL/well in 96-well plate in an incubator under a 95%O2–5%CO2 gas mixture, at 37°C for 48 hours. Two forms of stimulation were used to culture: no stimulation (none) and 100 μg/mL of Df allergen (Df). The concentrations of IFN-γ, IL-2, IL-4, IL-5, and IL-13 in the culture supernatants were determined by ELISA (Quantikine, R&D Systems Inc., Minneapolis, MN), using the procedures described in the respective instruction manuals.
5
Splenic and Aortic Cell Analysis in Atherosclerosis
6
Isolation of Lacrimal Gland Epithelial Cells
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Lacrimal glands were minced in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and 100 U/ml Penicillin Streptomycin (Pen Strep, Thermo Fisher Scientific), and digested at 37°C for 40 min with 1 mg/ml collagenase type I (Wako), 1 mg/ml hyaluronidase type I (Sigma), 0.01 mg/ml DNAse I (Roche), and 100 U/ml Pen Strep in DMEM. After being digested, they were filtered through a 70-μm nylon mesh, centrifuged, and rinsed twice with DMEM containing 10% FBS. Epithelial cells from the cell suspension were collected by positive selection using Miltenyi mouse CD326 (EpCAM) MicroBeads. We confirmed that purity was more than 80% (Supplementary Figure 5 ).
7
Isolation of Skin-Derived Immune Cells
8
Epithelial Cell Isolation from Mouse Tissues
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Tissues were minced and homogenized as described above and digested with 1 mg/mL collagenase type I (Wako), 1 mg/mL hyaluronidase type I (Sigma), and 0.01 mg/mL DNase I (Roche) in DMEM containing 10% FBS at 37 °C for 40 min. They were then filtered through a 70-μm nylon mesh, centrifuged (600× g, 4 °C, 5 min), rinsed twice with DMEM containing 10% FBS, and filtered through a 40-μm nylon mesh. Epithelial cells from the cell suspension were collected by positive selection using Miltenyi mouse CD326 (epithelial cell adhesion molecule; EpCAM) MicroBeads (130-105-958) [56 (link)]. We confirmed that purity was more than 80% (Figure S7 ). Total RNA was extracted as described above.
9
Isolation of Murine Vascular Smooth Muscle Cells
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VSMC cultures were established from male and female mice euthanized by CO2-inhalation at 6–8 weeks. The aorta was dissected and cleaned from adventitial tissue. Next, aortic vessels from ~4 individuals per genotype were pooled and cut into pieces (~1 mm) prior to a first enzymatic digestion step (Papain (0.7 mg/mL) from Sigma Aldrich (Schnelldorf, Germany) for 1 h at 37 °C. Following the second enzymatic digestion (Hyaluronidase Type 1 (1 mg/mL) and Collagenase Type 2 (1 mg/mL), both from Sigma Aldrich (Schnelldorf, Germany), under sterile conditions for 25 min at 37 °C. The isolated cells were then centrifuged (300×g for 7 min) and resuspended in DMEM Glutamax cell culture medium containing 5% PenStrep and 10% FCS. VSMC cultures were maintained in sterile incubators at 37 °C and 6% CO2 and received fresh medium every third day. After approximately 7 days, when cells grew to 80% confluency, subsequent in vitro experiments were performed.
10
Isolating Aortic Cells from Mice
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