Islets were isolated from NOR mice. Briefly, the pancreas was injected through the papilla of Vaters with 2.5 mL Collagenase XI solution: Collagenase XI (Sigma-Aldrich, #C7657) dissolved in HBSS buffer supplemented with Ca2+, Mg2+ (CarlRoth, #9119.1) and 0.08% BSA. The harvested pancreas was incubated in a water bath at 37 °C for 15 min. The digestion was stopped by the addition of cold RPMI medium (Corning (Corning, NY, USA), #10–040-CV) supplemented with 10% FBS. Following digestion, the pancreas was transferred through a metallic strainer and washed two times with RPMI. Islet separation was performed with 10 mL of Histopaque-1077 (Sigma-Aldrich, #10771) overlaid with 5 mL RPMI and centrifuged at 850× g for 15 min. Islets are transferred from the gradient with a pipette to a new tube with RPMI. Islets were washed three times with RPMI, resuspended in RPMI with 10% FBS and hand-picked. For treatments, islets were cultured in DMEM low glucose (PAN-Biotech), supplemented with 10% FBS and 1% penicillin/streptomycin, for the indicated time.
Collagenase xi
Manufactured by Merck Group
Sourced in United States, Canada, Germany, United Kingdom
Collagenase XI is a purified enzyme preparation derived from Clostridium histolyticum. It is used for the dissociation and dispersion of a variety of tissues, including connective tissue.
Automatically generated - may contain errors
Lab products found in correlation
Hyaluronidase, by Merck Group (77 mentions)
Collagenase 1, by Merck Group (54 mentions)
Dnase 1, by Merck Group (44 mentions)
Dnase, by Merck Group (20 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (17 mentions)
Dispase, by STEMCELL (15 mentions)
Dispase, by Thermo Fisher Scientific (14 mentions)
Type 4 bovine pancreatic dnase, by Merck Group (12 mentions)
Collagenase type 1, by Merck Group (12 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (12 mentions)
Dispase 2, by Merck Group (10 mentions)
Anti cd31 390, by BioLegend (8 mentions)
Penicillin, by Thermo Fisher Scientific (8 mentions)
Anti cd45.2 104, by BioLegend (7 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (7 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (7 mentions)
Rbc lysis buffer, by BioLegend (6 mentions)
Streptomycin, by Thermo Fisher Scientific (6 mentions)
Dnase 1, by Roche (6 mentions)
Percoll, by GE Healthcare (6 mentions)
221 protocols using collagenase xi
1
Isolation and Culture of Mouse Pancreatic Islets
2
Mouse Pancreas and Islet Isolation
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For pancreas isolations, dissected mouse pancreas was placed directly into 4% paraformaldehyde in PBS on ice followed by fixation overnight at 4 C and further processing for histology. For islet isolations, the common bile duct was perfused with 3 mL collagenase XI (1,000 units/ mL; Sigma-Aldrich) in Hanks' balanced salt solution, digested with collagenase XI and then filtered through a 40 mm filter. Handpicked islets were recovered overnight at 37 C in a 5% CO 2 humidified incubator or used immediately for experiments.
3
Tumor Immune Cell Profiling by Flow Cytometry
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Flow cytometry was carried out as previously described [11 (link)]. The mice were killed by cervical dislocation, and the tumor tissue was harvested, cut up, and incubated with 488 U/ml collagenase I (Sigma, #C0130), 230 U/ml collagenase Xi (Sigma, #C7657), 125 U/ml hyaluronidase (Sigma, #H3506), and 60 U/ml DNase I (Sigma, #d4527) at 37° C for 45 min. The digest was filtered by a 70 μm cell filter, and 1×107 cells were stained with an FITC-conjugated F4/80 antibody from Abcam and a PE-conjugated CD206 antibody from R&D Systems and analyzed by flow cytometry.
4
Intestinal Epithelial and Mesenchymal Cell Isolation
5
Isolation and Culture of Bovine MDSCs
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Skeletal muscle tissues were collected from newborn calves after obtaining approval from the Animal Welfare Committee of Northeast Agricultural University, Heilongjiang Province, China. Skeletal muscle tissues were pooled and finely minced. Subsequently, they were digested by treatment with 0.2% collagenase XI (Sigma-Aldrich, St. Louis, MO, USA) for 2 h, followed by treatment with 0.25% trypsin (Sigma) for 30 min. The isolated bovine MDSCs were cultured in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% FBS, 100 U/mL penicillin, and 100 μg/ml streptomycin at 37°C and 5% CO2 in a humidified atmosphere.
6
Characterizing Cardiac Immune Populations
7
Isolation of Wound-Infiltrating Immune Cells
8
Isolation and Analysis of Hepatic Immune Cells
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Hepatic cells were isolated according to a previously described method22 with minor modifications. To enumerate absolute number of immune cell subsets, mice were fully perfused with PBS to minimize contamination of peripheral blood, and livers were cut into same‐sized (same‐volume) segments. To enumerate absolute number of immune cell subsets, mouse liver was cut into same‐sized segments. Each segment was incubated with an enzyme mixture containing 675 U/mL collagenase I, 18.75 U/mL collagenase XI, and 9 U/mL hyaluronidase (Sigma‐Aldrich) in calcium‐ and magnesium‐containing Hanks’ balanced salt solution for 40 minutes at 37°C with gentle shaking. After digestion with enzyme mix, cells were washed and further processed by percoll (Roche) gradient to enrich CD45+ immune cells. Single‐cell suspensions were incubated in culture supernatant from the 2.42G2 hybridoma (Fc receptor block, ATCC HB‐197) prior to staining with the indicated antibodies (BioLegend). Stained cells were acquired using a BD LSRFortessa flow cytometer (BD Biosciences) and analyzed with FlowJo (Tree Star). Side‐scattered light (SSClow) and forward scattering light (FSClow) dead cells and doublet cells were gated out and analyzed with FlowJo.
9
Isolation of Mononuclear Liver Cells
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Mononuclear liver cells were isolated from the tumor non-affected area of the liver tissue as previously described (20 (link)). In brief, the tissue underwent a series of flushing steps to remove excess sinusoidal blood, followed by a three-step perfusion protocol in which the final step involved enzymatic processing (with collagenase XI, Sigma). Supernatant obtained through these steps was washed and layered onto the Ficoll-Hypaque media solution for the density gradient centrifugation to isolate leukocytes in the same way as PBMCs. Whole tonsils were mechanically processed by cutting and passing through a 100 μm strainer, followed by a 40 μm straining step, and finally a density gradient centrifugation in the same way as liver and blood samples. Post-isolation, cells from liver and tonsil were frozen in FBS supplemented with 10% DMSO and stored in liquid nitrogen, similar to PBMC.
10
Isolation and Culture of Bovine Nucleus Pulposus Cells
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NP cells were isolated from the caudal discs of 3 adult cows. The central NP tissue was dissected out and the three upper discs were pooled from each animal. Tissue was finely minced and digested overnight with collagenase XI (0.8 mg/mL (≥960 CDU/mL)) in DMEM/F12 medium (both from Sigma-Aldrich, St. Louis, USA) at 37°C. Cells were then maintained in culture at 5000 cells/cm2 in DMEM/F12 culture medium (Life Technologies, Paisley, UK) routinely supplemented with 10% (v/v) foetal bovine serum (FBS) (PAA, Yeovil, UK), 50 μg/mL ascorbic acid (Sigma-Aldrich), 0.5% (v/v) gentamicin (Life Technologies), and 0.05% fungizone (Life Technologies), at 37°C in a humidified atmosphere of 5% CO2. Cells were passaged as standard by trypsinisation upon reaching 80% confluence and used at passage 4 or 5.
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