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Elastase

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Elastase is a proteolytic enzyme that cleaves peptide bonds in proteins, specifically at the carboxyl side of small aliphatic amino acid residues such as alanine, valine, serine, and threonine. It is used in various laboratory applications, including the study of protein structure and function.

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Lab products found in correlation

Anti s100a4, by Abcam (2 mentions) Anti nk1, by BioLegend (2 mentions) Anti f4 80, by BioLegend (2 mentions) Anti cd31, by BioLegend (2 mentions) Anti cd3, by BioLegend (2 mentions) Anti sp c, by ABclonal (2 mentions) Anti cd45, by BioLegend (2 mentions) Anti epcam, by BioLegend (2 mentions) Anti ly6g, by BioLegend (2 mentions) Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (2 mentions) Hanks balanced salt solution (hbss), by Roche (2 mentions) Liberase, by Roche (2 mentions) Dnaase, by Merck Group (1 mentions) Rbc lysis buffer, by BD (1 mentions) Cell staining buffer, by BioLegend (1 mentions) Dnase 1, by Worthington (1 mentions) Anti cd19, by BioLegend (1 mentions) Elastase, by Merck Group (1 mentions) Collagenase type 2, by Thermo Fisher Scientific (1 mentions) Type 2 collagenase, by Roche (1 mentions)

13 protocols using elastase

1

Isolation of Human ATII Cells from Donor Lung

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Human ATII cells were isolated from a donor lung (The Gift of Life Donor Program, Philadelphia, PA) by modification of previously published protocols (22 (link), 33 (link), 34 (link)). All procedures were approved by the Penn State College of Medicine Institutional Review Board. The lung was lavaged, instilled with 12.9U/ml elastase (Roche, Indianapolis, IN), and incubated at 37°C for 50min. The tissue was homogeneized, filtered, and treated with DNAase (Sigma, St. Louis, MO). Cell depletion was performed with anti-CD-14 magnetic beads (Life technologies, Carlsbad, CA) and by incubation with IgG (Sigma) on petri dishes, followed by treatment with RBC lysis buffer (BD Biosciences, San Jose, CA). ATII cells were suspended in DMEM containing 10% FBS, 2mM glutamine, 2.5μg/ml amphotericin B, 100μg/ml streptomycin, penicillin G and gentamicin (D10). Cell purity was confirmed by Papanicolaou stain and electron microscopy (35 (link)). The lung donor was a 20 year old male with the SP-A1/SP-A2 haplotype 6A26A3/1A01A1.
Silveyra P., Chroneos Z.C., DiAngelo S.L., Thomas N.J., Noutsios G.T., Tsotakos N., Howrlylak J.A., Umstead T.M, & Floros J. (2014). Knockdown of Drosha in human alveolar type II cells alters expression of SP-A in culture: a pilot study. Experimental lung research, 40(7), 354-366.
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2

Isolation of Single-Cell Suspension from Mouse Lungs

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Lungs were perfused with 20 mL PBS followed by the inflation with 2 mL sterile protease cocktail containing collagenase/dispase (cat. no. 11097113001, Roche, Switzerland), elastase (cat. no. LS002279, Worthington Biochemical, NJ), and DNaseI (cat. no. 10104159001, Roche) in DMEM/F12 medium. Lungs were disrupted with scissors and incubated with the protease cocktail in a warm room for 45 min on a shaking platform. The protease digestion was halted by adding 8 mL DMEM/F12 medium containing 10% FBS. The dissociated lung was pelleted by centrifugation. Cell pellets were washed with PBS, resuspended in 2 mL ACK red cell lysis buffer (NH4Cl, KHCO3, EDTA disodium, pH 7.4) and incubated at room temperature for 2 min. Five milliliters of DMEM/F12 containing 10% FBS was added to the cell suspension, which was then filtered through a 40-μm cell strainer to obtain a single-cell suspension. After spin down to remove the lysis buffer, cells were resuspended in a cell-staining buffer (BioLegend, cat. no. 420201) containing antibody cocktails. Cell number was counted and cell suspension was dilute as 1–2 × 107 cells/mL. Cells were then incubated with the antibody cocktail on ice for 20 min to 1 h, washed twice with FACS staining buffer and subjected to FACS analysis and sorting.
Chen H., Durinck S., Patel H., Foreman O., Mesh K., Eastham J., Caothien R., Newman R.J., Roose-Girma M., Darmanis S., Warming S., Lattanzi A., Liang Y, & Haley B. (2022). Population-wide gene disruption in the murine lung epithelium via AAV-mediated delivery of CRISPR-Cas9 components. Molecular Therapy. Methods & Clinical Development, 27, 431-449.
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3

Isolation of Murine Lung Cell Subsets

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BALB/c mice were perfused with 5 ml of HBSS through the pulmonary artery via the right ventricle and instilled with 4.5 U/ml of elastase (Roche Diagnostics) via tracheal cannula. The lung lobes were then minced in 100 U/ml DNase I (Aladdin). Cells in suspension were subsequently filtered through a 40 μm nylon mesh and then sorted by FACS. Anti-CD45 (BioLegend, 157607, 1:200 dilution) and anti-CD3 (BioLegend, 100209, 1:200 dilution) antibodies were used for the isolation of T cells. Anti-CD45 and anti-CD19 (BioLegend, 152407, 1:200 dilution) antibodi were used for isolation of B cells. Anti-CD45 and anti-NK-1.1 (BioLegend, 108703, 1:200 dilution) antibody were used for isolation of NK cells. Anti-CD45 and anti-F4/80 (BioLegend, 123105, 1:200 dilution) antibodies were used for the isolation of macrophages. Anti-CD45 and anti-Ly6G (BioLegend, 127627, 1:200 dilution) antibodies were used for the isolation of neutrophils. Anti-EpCAM (BioLegend, 324215, 1:200 dilution) and anti-SP-C (abclonal, A1835, 1:500 dilution) antibodies were used for the isolation of alveolar epithelial cells. anti-CD31 (BioLegend, 102409, 1:200 dilution) antibody was used for the isolation of endothelial cells. Anti-S100A4 (abcam, ab197896, 1:500 dilution) antibody was used for the isolation of fibroblasts.
Zeng Z., Xu S., Wang F., Peng X., Zhang W., Zhan Y., Ding Y., Liu Z, & Liang L. (2022). HAO1-mediated oxalate metabolism promotes lung pre-metastatic niche formation by inducing neutrophil extracellular traps. Oncogene, 41(29), 3719-3731.
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4

Purification of Murine Neutrophils and Alveolar Epithelial Cells

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For isolation of neutrophils, bone marrow cells derived from 6 to 8-week-old BALB/c mice were harvested in Hank’s buffered salt solution (HBSS; Gibco), subsequently filtered through 40-μm nylon mesh and added to the top of a 2-layer percoll (GE Healthcare) gradient (72% and 63.5% in PBS), followed by centrifugation at 810 g for 20 min at 4 °C. Neutrophils enriched in the interface of 63.5%-72% fractions were confirmed to be of >95% purity by immunofluorescence (Supplementary figure S4F). For isolation of alveolar epithelial cells, BALB/c mice were perfused with 5 ml of HBSS through the pulmonary artery via the right ventricle and instilled with 4.5 U/ml of elastase (Roche Diagnostics) via tracheal cannula. The lung lobes were then minced in 100 U/ml DNase I (Aladdin). Cells in suspension were subsequently filtered through 40 μm nylon mesh and then enriched by IgG panning. The purified alveolar epithelial cells were confirmed to be of >90% purity by immunofluorescence (Supplementary figure 1J).
Zeng Z., Xu S., Wang F., Peng X., Zhang W., Zhan Y., Ding Y., Liu Z, & Liang L. (2022). HAO1-mediated oxalate metabolism promotes lung pre-metastatic niche formation by inducing neutrophil extracellular traps. Oncogene, 41(29), 3719-3731.
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5

Isolation of Murine Lung Cell Populations

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BALB/c mice were perfused with 5 ml of HBSS through the pulmonary artery via the right ventricle and instilled with 4.5 U/ml of elastase (Roche Diagnostics) via tracheal cannula. The lung lobes were then minced in 100 U/ml DNase I (Aladdin). Cells in suspension were subsequently filtered through a 40 μm nylon mesh and then sorted by FACS. Anti-CD45 (BioLegend, 157607, 1:200 dilution) and anti-CD3 (BioLegend, 100209, 1:200 dilution) antibodies were used for the isolation of T cells. Anti-CD45 and anti-CD19 (BioLegend, 152407, 1:200 dilution) antibodi were used for isolation of B cells. Anti-CD45 and anti-NK-1.1 (BioLegend, 108703, 1:200 dilution) antibody were used for isolation of NK cells. Anti-CD45 and anti-F4/80 (BioLegend, 123105, 1:200 dilution) antibodies were used for the isolation of macrophages. Anti-CD45 and anti-Ly6G (BioLegend, 127627, 1:200 dilution) antibodies were used for the isolation of neutrophils. Anti-EpCAM (BioLegend, 324215, 1:200 dilution) and anti-SP-C (abclonal, A1835, 1:500 dilution) antibodies were used for the isolation of alveolar epithelial cells. anti-CD31 (BioLegend, 102409, 1:200 dilution) antibody was used for the isolation of endothelial cells. Anti-S100A4 (abcam, ab197896, 1:500 dilution) antibody was used for the isolation of fibroblasts.
Zeng Z., Xu S., Wang F., Peng X., Zhang W., Zhan Y., Ding Y., Liu Z, & Liang L. (2022). HAO1-mediated oxalate metabolism promotes lung pre-metastatic niche formation by inducing neutrophil extracellular traps. Oncogene, 41(29), 3719-3731.
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6

Enzymatic Digestion and Isolation of Cardiac Cells

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Five different protocols for enzymatic digestion and cell isolation of cardiac tissue was assessed in murine and human heart tissue, based on previous protocols applied to mouse tissue (Farbehi et al., 2019 ; Gladka et al., 2018 (link)) and one which had been used on human tissue (Bajpai et al., 2018 (link)). Dissected tissue was placed in a tube and incubated with one of the following: (1) HBSS with 1 mg/ml Collagenase Type II (ThermoFisher Scientific, Waltham, MA, USA) and 3 mM CaCl2, (2) HBSS with 0.5 mg/ml Elastase (Sigma Aldrich, St Louis, MO, USA) and 3 mM CaCl2, (3) HBSS with 1 mg/ml Collagenase Type II and 0.5 mg/ml Elastase, (4) DMEM with 450 U/ml Collagenase Type I and 60 U/ml Hyaluronidase or (5) DMEM with Liberase (Roche, Basel, Switzerland). Tissue was digested for 2 h at 37 °C in protocol 1–3 and 1 h at 37 °C in protocol 4–5. Mouse tissue was digested for 20 min in all protocols. After incubation, cells were passed through 100 or 200 μM cell strainers, washed with HBSS and resuspended in medium for primary cells.
Pimpalwar N., Czuba T., Smith M.L., Nilsson J., Gidlöf O, & Smith J.G. (2020). Methods for isolation and transcriptional profiling of individual cells from the human heart. Heliyon, 6(12), e05810.
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7

Isolation of Murine Neutrophils and Alveolar Epithelial Cells

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For isolation of neutrophils, bone marrow cells derived from 6 to 8-week-old BALB/c mice were harvested in Hank’s buffered salt solution (HBSS; Gibco), subsequently filtered through 40-μm nylon mesh and added to the top of a 2-layer Percoll (GE Healthcare) gradient (72% and 63.5% in PBS), followed by centrifugation at 810 g for 20 min at 4 °C. Neutrophils enriched in the interface of 63.5–72% fractions were confirmed to be of >95% purity by immunofluorescence (Supplementary Fig. S4F). For isolation of alveolar epithelial cells, BALB/c mice were perfused with 5 ml of HBSS through the pulmonary artery via the right ventricle and instilled with 4.5 U/ml of elastase (Roche Diagnostics) via tracheal cannula. The lung lobes were then minced in 100 U/ml DNase I (Aladdin). Cells in suspension were subsequently filtered through 40 μm nylon mesh and then enriched by IgG panning. The purified alveolar epithelial cells were confirmed to be of >90% purity by immunofluorescence (Supplementary Fig. 1J).
Zeng Z., Xu S., Wang F., Peng X., Zhang W., Zhan Y., Ding Y., Liu Z, & Liang L. (2022). HAO1-mediated oxalate metabolism promotes lung pre-metastatic niche formation by inducing neutrophil extracellular traps. Oncogene, 41(29), 3719-3731.
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8

Isolating Mouse Taste Cells

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For isolation of taste buds or individual cells, 8–10 weeks old C57Bl6 mice were euthanized with CO2followed by cervical dislocation before tongues were removed. Taste cells were isolated from mouse (NMRI, 6–8-week old)
circumvallate (CV) papilla. A tongue was injected between the epithelial and muscle layers with 0.7 mg/ml collagenase B, 1 mg/ml
dispase II, 0.2 mg/ml elastase (all from Roche Diagnostics), and 0.5 mg/ml trypsin inhibitor (Sigma-Aldrich) dissolved in a
solution (mM): 140 NaCl, 20 KCl, 0.3 MgCl2, 0.3 CaCl2, 10 HEPES-NaOH (pH 7.4). The tongue was incubated in
an oxygenated Ca-free solution (in mM): 120 NaCl, 20 KCl, 1 MgCl2, 0.5 EGTA, 0.5 EDTA, 10 HEPES-NaOH (pH 7.4) for
20–30 min. The epithelium was then peeled off from the underlying muscle, pinned serosal side up in a dish covered with
Sylgard resin, and incubated in the Ca-free solution for 10–30 min. The isolated epithelium was kept at room temperature
in a solution (mM): 130 NaCl, 10 NaHCO3, 5 KCl, 1 MgCl2, 1 CaCl2, 10 HEPES-NaOH (pH 7.4), 5
glucose, 2 Na-pyruvate). Taste cells were removed from the CV papilla by gentle suction with a firepolished pipette with an
opening of 70–90 μm and then expelled into an electrophysiological chamber.
Romanov R.A., Lasher R.S., High B., Savidge L.E., Lawson A., Rogachevskaja O.A., Zhao H., Rogachevsky V.V., Bystrova M.F., Churbanov G.D., Adameyko I., Harkany T., Yang R., Kidd G.J., Marambaud P., Kinnamon J.C., Kolesnikov S.S, & Finger T.E. (2018). Chemical synapses without synaptic vesicles: purinergic neurotransmission via a CALHM1 channel-mitochondrial signaling complex. Science signaling, 11(529), eaao1815.
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9

Characterizing SpyCas9 Conformational Changes

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Limited proteolysis experiment was conducted at room temperature. SpyCas9 with or without AcrIIA4 and the purified SpyCas9-sgRNA complex in the absence or presence of AcrIIA4 were mixed with Elastase (Roche) at a 1:120 (w/w) ratio and incubated at room temperature. Aliquots were taken at noted time points and immediately quenched by the addition of equal amount of 2× SDS–polyacrylamide gel electrophoresis loading dye (Bio-Rad). Samples were further boiled for 5 min at 95°C and then resolved by 4 to 20% Tris-Glycine Mini Gels (Bio-Rad).
Shin J., Jiang F., Liu J.J., Bray N.L., Rauch B.J., Baik S.H., Nogales E., Bondy-Denomy J., Corn J.E, & Doudna J.A. (2017). Disabling Cas9 by an anti-CRISPR DNA mimic. Science Advances, 3(7), e1701620.
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10

4T1 Mammary Tumor Metastasis Assay

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BALB/c mice were inoculated orthotopically in the mammary fat pad with 104 4T1 cells per mouse. Mice (18 per group) were treated daily with PBS or with PCPA [10 mg/kg, intraperitoneally (i.p.), 5 days a week] for 28 days. Primary tumor growth was monitored once a week with a caliper, and the volume was then calculated using the formula d2 × D/2, where d and D are the short and the long diameters, respectively. For evaluation of lung micrometastases, clonogenic assays were performed on day 28: lungs were removed from each mouse, minced, digested in 5 ml of an enzyme mix containing 1 × Hank's balanced salt solution (Lonza, Basel, Switzerland), 1 mg/ml collagenase type IV (Worthington, Lakewood, NJ), and 6 U/ml elastase (Roche), and incubated for 75 minutes at 4°C on a platform rocker. After incubation, samples were filtered through and plated at different dilutions (1:2, 1:10, and 1:100) onto 10-cm tissue culture dishes in DMEM–10% FBS containing 10 μg/ml thioguanine (Sigma, Saint Louis, MO) for clonogenic growth. After 14 days of incubation at 37°C, tumor cells, which are thioguanine-resistant, formed individual colonies representing micrometastases that were fixed with methanol and stained with Giemsa (Sigma). Lung metastases were also evaluated on formalin-fixed paraffin-embedded sections stained by hematoxylin-eosin, by two operators, blindly.
Ferrari-Amorotti G., Chiodoni C., Shen F., Cattelani S., Soliera A.R., Manzotti G., Grisendi G., Dominici M., Rivasi F., Colombo M.P., Fatatis A, & Calabretta B. (2014). Suppression of Invasion and Metastasis of Triple-Negative Breast Cancer Lines by Pharmacological or Genetic Inhibition of Slug Activity. Neoplasia (New York, N.Y.), 16(12), 1047-1058.
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