Mice were sacrificed at time points indicated by intraperitoneal (i.p.) injection of sodium pentobarbital. The lungs were then mechanically disrupted by GentleMACS dissociation (Miltenyi Biotec) (Lung program 01_01) in 3 ml RPMI 1640 (GIBCO) containing 20 μg/ml Liberase and 10 U/ml Dnase (Roche), followed by digestion for 30 min at 37°C and final GentleMACS homogenization (Lung program 02_01). Single cell suspensions were prepared by digestion in collagenase/Dnase (Roche) solution; lungs were digested for 30 minutes at 37°C, MLNs were digested 15 minutes at 37°C. Next, the cell suspension was passed through a 100 μm filter and red blood cells were lysed using ammonium chloride lysis buffer (10 mM KHCO3, 155 mM NH4Cl, 0,1 mM EDTA in MilliQ water). Auricular LNs were digested with 100μg/ml Liberase TL and 100μg/ml Dnase I (Roche) for 30 minutes at 37°C before passing through a 70 μm cell strainer.
Collagenase dnase
Manufactured by Roche
Collagenase/DNase is a laboratory reagent used to dissociate tissues and cell clusters. It contains a mix of enzymes, including collagenase and deoxyribonuclease (DNase), that work together to break down the extracellular matrix and release individual cells from the tissue.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Liberase tl, by Roche (1 mentions)
Liberase, by Roche (1 mentions)
Dnase 1, by Roche (1 mentions)
Gentlemacs, by Miltenyi Biotec (1 mentions)
Gentlemacs, by Roche (1 mentions)
Dnase, by Roche (1 mentions)
Percoll, by GE Healthcare (1 mentions)
Red blood cell lysis, by Merck Group (1 mentions)
70 m cell strainer, by BD (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Cd4 apc, by Thermo Fisher Scientific (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Golgistop, by BD (1 mentions)
Cd11b, by BD (1 mentions)
4 protocols using collagenase dnase
1
Lung and Lymph Node Dissociation
2
Isolating Immune Cells from Murine Tissues
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When indicated, the spleen, liver, lung, and tumor were collectedfrom mice and incubated with 0.1 mg collagenase/DNase (Roche) for 30 min at 37 °C. Single-cell suspensions were prepared by mechanically disrupting the organs through a 70 µm cell strainer (Falcon). Tumor-infiltrating mononuclear cells, liver cells, and lung cells were isolated by centrifugation with 44% Percoll (GE Healthcare). Cells were then subjected to red blood cell lysis (Sigma-Aldrich). Blood was collected in PBS containing 2% FCS, 0.1% sodium azide, and 2.5 U/ml heparin. Peripheral blood mononuclear cells (PBMCs) were prepared by lysing erythrocytes with red blood cell lysis buffer. Mononuclear cells from the abovementioned organs were washed, resuspended in RPMI + 2% FCS (Gibco), counted and kept on ice until further analysis.
3
Multiparametric Flow Cytometry Analysis of Lung Cells
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Total lung tissue was digested (37 degree C, 45 minutes) with 5 mg/ml DNase/collagenase (Roche Diagnostics) and a single cell suspension was obtained by mechanical dissociation of the tissue. The cells were counted using a trypan blue exclusion assay and 106 total lung cells from each mouse were used for the flow cytometry assay. Total lung cells were stained with the surface antibody CD4 APC (eBioscience). Prior to intracellular staining the cells were stimulated with 50 ng/ml PMA (Sigma-Aldrich), 1 μg/ml Ionomycin (Sigma-Aldrich) and 1 μl/ml Golgi Stop (BD Biosciences). After permeabilization with Cytofix/Cytoperm (BD Biosciences), staining with intracellular antibodies (eBioscience) against Ifnγ, Il13 and Il17 was performed. Cell acquisition was completed using LSRII cytometer.
4
Isolation of Splenic Dendritic Cells
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DCs from spleen were isolated using a previously described method (86 (link)). In brief, spleens were digested using DNase/Collagenase (Roche Diagnostics, Basel, Switzerland/Worthington Biochemical Corporation, Lakewood, New Jersey) at room temperature (RT) for 20 min and filtered to create a single cell suspension. Light density cells were isolated by centrifugation in a 1.077 g/cm3 NycoPrep™ medium (AXIS Shield PoC AS, Dundee, Scotland). Negative selection using a rat monoclonal Ab cocktail against CD3-ϵ (KT3-1.1), Thy-1 (T24/31.7), Ly6G/Ly6C (1A8), CD19 (ID3) and erythrocytes (TER-119) together with anti-rat immunoglobulin immunomagnetic beads (Qiagen, Hilden, Germany) was used to isolate DCs. For sorted DCs, these isolated cells were then labelled with the following fluorophore-conjugated mAbs: CD11c (N418), CD45RA (14.8), CD8 (53-6.7), CD11b (M1/70), CD49b (DX5) (Becton Dickinson, Franklin Lakes, New Jersey; eBioscience, San Diego, California; BioLegend, San Diego, California; TONBO, San Diego, California) and sorted on BD Influx machine according to gating strategy provided (Supplementary Figure 4A
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