Mice were sacrificed by intraperitoneal injection of Fatal-Plus solution (Vortech), and lungs were perfused with 10 ml of sterile normal saline via the pulmonary artery. The airway was cannulated via tracheostomy with a 20-gauge metallic angiocatheter, and 3 ml of dispase (50 caseinolytic units/ml, Corning) was instilled, followed by 0.5 ml of 1% low-melt agarose (warmed to 45°C). Lungs were rapidly cooled on ice for 2 min, incubated in 1 ml of dispase for 45 min at room temperature, and transferred to a culture dish containing deoxyribonuclease I (100 U/ml) (Worthington). The parenchymal lung tissue was gently teased from the bronchi and homogenized. Cell suspensions were filtered, collected by centrifugation, and placed on prewashed 100-mm tissue culture plates coated with CD45 and CD32/16 antibodies (BD Biosciences). After incubation for 60 min at 37°C in a 5% CO2 atmosphere to promote adherence of contaminating macrophages and fibroblasts, the AECII were gently panned from the plate, collected by centrifugation, and counted. For the Npt2b−/− animals, differential centrifugation was used to separate microliths from the cells. Cell viability determined with trypan blue staining was routinely >90%, and cell purity determined by SP-C staining ranged from 75 to 90%.
Dispase
Manufactured by Worthington
Sourced in United States
Dispase is a proteolytic enzyme that can be used for the dissociation of cells from a variety of tissues. It is a neutral protease that can effectively disrupt the extracellular matrix, making it useful for cell isolation and tissue dissociation applications.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Merck Group (17 mentions)
Collagenase, by Merck Group (10 mentions)
Collagenase d, by Roche (10 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Dnase 1, by Worthington (7 mentions)
Collagenase 3, by Worthington (5 mentions)
Dnase, by Worthington (5 mentions)
Myelin removal beads, by Miltenyi Biotec (5 mentions)
Collagenase type 2, by Worthington (5 mentions)
Collagenase 2, by Roche (5 mentions)
Percoll, by GE Healthcare (5 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (5 mentions)
Collagenase type 1, by Worthington (4 mentions)
Collagenase, by Worthington (4 mentions)
Streptomycin, by Merck Group (4 mentions)
Penicillin, by Merck Group (4 mentions)
Collagenase 4, by Worthington (4 mentions)
Dnase, by Merck Group (4 mentions)
Collagenase, by Roche (4 mentions)
Papain, by Worthington (4 mentions)
90 protocols using dispase
1
Isolation and Purification of Alveolar Epithelial Type II Cells
2
Isolation and Culture of Rat Mesenchymal Stem Cells
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HEK-293T cell line was purchased from ATCC. Rat MSC were isolated from subcutaneous adipose tissue. Briefly, the tissue sample was fragmented under sterile conditions to a homogeneous mass and subjected to enzymatic treatment in Dulbecco’s modified Eagle’s medium (DMEM) medium (Gibco, Waltham, MA, USA) with 10 U/mL dispase (Worthington Biochemical, Lakewood, NJ, USA) and 200 U/mL type I collagenase (Worthington Biochemical, Lakewood, NJ, USA) at 37 °C for 30 min. After centrifugation for 10 min at 200× g the cell pellet was resuspended in DMEM and filtered through 40 μm nylon membrane. Isolated cells were maintained in 4.5 g/L D-glucose DMEM with 10% fetal bovine serum (FBS, Gibco, Waltham, MA, USA) and 1% penicillin/streptomycin solution at 37 °C and 5% CO2. For all experimental procedures, including CS generation and preliminary testing, early passage cells were used (P3–P4). The cells were passaged by Trypsin-Versene 1:1 solution (Paneco, Moscow, Russia).
3
Isolation of Epidermal Mononuclear Cells
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Abdominal skin was collected immediately after surgery and processed as previously described [35 (link)]. Briefly, skin pieces were removed from underlying tissue using a skin graft knife (Swann-Morton, UK), and the resulting skin grafts were passed through a skin graft mesher (Zimmer Biomet, USA). To facilitate the mechanical separation of the epidermis from underlying dermis, the meshed skin pieces were placed in RPMI 1640 (Lonza) containing 0.14 U/mL dispase (Worthington Industries, USA) and 50 μg/mL gentamicin, and rotated at 4°C overnight. The skin was then washed in PBS and epidermis was separated from dermis using fine forceps. To liberate epidermal cells, epidermal tissue was incubated with RPMI 1640 containing 200 U/mL collagenase Type IV (Worthington) at 37°C for 120 min. A tea strainer was used to separate the cells from undigested tissue, and then the supernatants were passed through a 100 μm cell strainer (Greiner Bio-One, Germany). The epidermal cells were pelleted, washed twice with cold PBS and resuspended in RPMI 1640 for centrifugation on a Ficoll-Paque PLUS (GE Healthcare, USA) density gradient at 450 x g for 20 min to harvest the epidermal MNPs. For Caspase 3 experiments, a Dead Cell Removal kit (StemCell Technologies, Canada), was used as per manufacturer’s directions to remove dead cells prior to culture.
4
Isolation and Purification of Murine Type II AECs
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Primary murine type II AEC were isolated and purified using a modification of published methods (Corti et al. 1996 (link); Mendez et al. 2006 (link); Sturrock et al. 2014 (link)). Briefly, lung cells were released by intratracheal instillation of neutral protease (Dispase; Worthington Biochemical Corp, Lakewood, NJ). The cell suspension was subjected to sequential filtration and leukocytes were removed by positive selection with anti-CD32 and anti-CD 45 (BD Bioscience, San Jose, CA). Mesenchymal cells were removed from the cell suspension by overnight adherence to tissue culture-treated plastic (=day 0). Primary AEC were allowed to attach on dishes coated with fibronectin for 48 h, after which nonadherent cells were removed by gentle washing. Purity of the epithelial cell preparations was confirmed by staining with murine antivimentin to identify mesenchymal and bone-marrow-derived cells. Routinely, fibroblast contamination was 3–6% on day 3 after isolation.
5
Isolation and Culture of Urothelial Cells
6
Flow Cytometry Profiling of Lung Immune Cells
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The same two lobes of the right lung were harvested from each A/J mouse (n = 6/group) for flow cytometry and incubated in digestion media consisting of collagenase (300 U/ml, Sigma), dispase (1 U/ml, Worthington), and DNAse (2 U/ml, Calbiochem) for 30 min at 37 °C with stirring. Cells were then passed through a 40 µm cell strainer (BD Falcon), and red blood cells eliminated with lysis solution. Single cells were resuspended in a solution of Brilliant buffer (BD Bioscience) and stained for 30 min at 4 °C with the following antibodies: CD45-Brilliant violet 510 (30F11), CD24-Brilliant violet 604 (M1/69), CD64-Brilliant violet 711 (X54-5/7.1), AI/AE- Brilliant violet 650 (M5/114.15.2), CD8-Alexafluor 700 (53–6.7), CD4-FITC (GK1.5), LyC6/LyG6-APC (rb6-8C5), CD11b-PE-Cy7 (M1/70), CD11c-PE-Cy5 (N418), CD206- Brilliant violet 421 (C068C2), CD69- Brilliant violet 785 (41.2F3), CD25-PE (3C7), CD107a-Alexafluor 647 (1D4B), and 5 μg/ml anti-mouse CD16/CD32 antibody (Biolegend) to reduce antibody binding to Fc receptors. Zombie NIR staining was used to exclude dead cells. Cells were analyzed using a Cytek Aurora equipped with 5 lasers (UV 355 nm, violet 405 nm, blue 488 nm, yellow-green 561 nm, and red 640 nm) and FlowJo x.10.0.7r2 software (Tree Star). The gating strategy used and shown in Supplemental Fig. 6 was adapted from a published protocol78 (link).
7
Pancreatic and Splenic Cell Isolation and Analysis
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One third of the pancreas and spleen removed from KC mice was minced and incubated separately in digestion media consisting of collagenase (300 U/ml, Sigma), dispase (1 U/ml, Worthington), and DNAse (2 U/ml, Calbiochem) for 30 minutes at 37 °C with stirring. Cells were then passed through a 40 µm cell strainer (BD Falcon), and red blood cells eliminated with lysing solution. Single cells were resuspended in a solution of PBS/0.5% BSA/0.1% azide and stained for 30 minutes at 4 °C with the following antibodies: CD45-VioGreen (30F11, Miltenyi), Gr-1-PE (RB6-8C5, Miltenyi), CD11b-FITC (M1/70.15.11.5, Miltenyi), CD19-APC (1D3/CD19, Biolegend), B220-PerCP-Cy5.5 (RA3-6B2, Biolegend), CD3-PE (145-2C11, Biolegend), CD4-FITC (Gk1.5, Miltenyi), CD8-APC (53–6.7, Biolegend) and 5 μg/ml anti-mouse CD16/CD32 antibody (Biolegend) to reduce antibody binding to Fc receptors. Propidium iodide staining was used to exclude dead cells. Cells were analyzed using an LSR II-DIVA 6.2 software (BD) with three laser sources (488 nm, 633 nm, 407 nm) and FlowJo x.10.0.7r2 software (Tree Star).
8
Murine Type II Alveolar Epithelial Cell Isolation
9
Mammary Gland Cell Isolation
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Number three and number four mammary fat pads were dissected and minced until no piece was larger than the bore of a 10 mL serological pipet. For every complete set of glands, 3 mL of the DMEM/F12 (Corning 10–092-CV) + 300 units/ml Collagenase 3 (Worthington LS004182) + 10 units/ml DNase I (Worthington LS002139) + 5 μM Y-27632 (Caymen Chemical 10005583) was used. This mixture was incubated for 2 hours rocking at 37°C. After primary digestion the epithelial pellet was washed with 1x PBS, RBCs were lysed, and RBC lysis buffer was removed. 1 mL of 0.05% Trypsin-EDTA (ThermoFisher 25300054) was then added (for up to three animals), mixed, and incubated for 5 minutes at 37°C. Trypsinization was halted with serum containing media, the sample was triturated, and the cells were pelleted again. This final pellet was incubated 1 mL of DMEM/F12 with 1 U/ml Dispase (Worthington LS02109) with 100 U/ml DNase (Worthington LS002139) at 37°C for five minutes. Dispase was halted by dilution and the final suspension was passed through a 40-micron filter.
10
Mammary Gland Cell Isolation
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