After rinsing with phosphate-buffered saline (PBS) and minced into 1 × 1 × 1 mm3 pieces, the specimens were digested with collagenase I (2 mg/ml, BioFroxx, Germany) for 60–90 min at 37 °C, 160 rpm. Then the mixture was filtered through 100 μm and 40 μm cell strainers successively, followed by resuspending the cell in DMEM/F12 medium containing 10% fetal bovine serum (BI, Israel) and incubating at 37 °C, 5% CO2. Antibody against vimentin (1:100, 5741, CST, USA) was used to identify the purity of primary stromal cells for its widely expressing in mesenchymally derived cells. The purity of isolated primary cells (> 95%) was identified by immunofluorescence using vimentin.
Collagenase 1
Manufactured by neoFroxx
Sourced in Germany, China, United States
Collagenase I is a type of enzyme used in laboratory settings. It is primarily used to break down collagen, a structural protein found in various tissues. The core function of Collagenase I is to facilitate the dissociation and isolation of cells from collagen-rich tissues, enabling researchers to study and work with those cells in controlled environments.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase 4, by neoFroxx (3 mentions)
Hyaluronidase grade 1, by neoFroxx (3 mentions)
Penicillin streptomycin, by Beyotime (1 mentions)
Trypsin, by Beyotime (1 mentions)
Cell incubator, by Thermo Fisher Scientific (1 mentions)
Mouse tumor infiltrating lymphocyte isolation kit, by Solarbio (1 mentions)
Dnase 1, by Tiangen Biotech (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Tgf β, by Thermo Fisher Scientific (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Il 10, by Thermo Fisher Scientific (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Il 1β, by Thermo Fisher Scientific (1 mentions)
Trifluoroacetic acid, by Sinopharm (1 mentions)
Ly2109761, by Selleck Chemicals (1 mentions)
Tgf β1, by GenScript (1 mentions)
7 protocols using collagenase 1
1
Isolation of Primary Stromal Cells
2
Isolation and Culture of Cardiac Fibroblasts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
One- to three-day-old neonatal Sprague–Dawley rats were obtained from the Experimental Animal Center of Harbin Medical University. The hearts were finely minced into small pieces (Golden et al., 2012 (link)). Heart tissues were digested with trypsin (Beyotime, China) and collagenase I (Biofroxx, Germany) into single cells. The cell suspension was mixed with a 1:2 volume of low-glucose DMEM (Hyclone, USA) containing 10% fetal bovine serum (BI, Israel) and 1% penicillin–streptomycin (Beyotime, China) to terminate digestion. Cardiac fibroblasts (CFs) were obtained by 90-min differential adherence and removal of non-adherent cells. Cell cultures were incubated in a 5% CO2 and 95% humidified incubator at 37°C. When the growth density of CFs was above 80%, the passage could be carried out. One or two generations of isolated fibroblasts were used for subsequent experiments. Normal glucose (NG, 5.5 mM) mediums were used in culturing CFs, while high-glucose (HG, 33 mM) culture conditions were used to mimic in vivo DCM.
3
Isolation and Characterization of Tumor-Infiltrating Lymphocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After the tumor-bearing mice were sacrificed, subcutaneous tumors were isolated completely. Weigh 0.5 g of tumor tissues were cut and then added to 3 mL of prewarmed (37 °C) 1640 medium containing 5% FBS (Gibco, USA), 1 mg/mL collagenase I (Biofroxx, Germany), and 0.02 mg/mL DNase I (TIANGEN, China). Tumor tissues were digested for 35 min in cell incubators (Thermo Scientific, USA, 37 °C, 5% CO2). The digested tissues were ground and filtered through 70-μm cell filters. A Mouse Tumor Infiltrating Lymphocyte Isolation Kit (Solarbio, China) was used to obtain TILs. Cell staining was performed as described above. The gating strategy for the flow cytometric analysis of immune cells in tumor tissue is shown in Additional file 1 : Fig. S3.
4
Measuring Colonic MPO Activity in Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To examine MPO activity, the colon tissues of mice were harvested post treatment and homogenized in cold PBS buffer with digestive enzymes (Collagenase I (BioFroxx, cat. no. 1904GR001), Collagenase IV (BioFroxx, cat. no. 2091GR001), Hyaluronidase Grade I (BioFroxx, cat. no. 1141GR001)). Then, the MPO activity of colon tissues was detected by a myeloperoxidase (MPO) activity assay kit (Nanjin Jiancheng, cat. no. A044-1-1) following the manufacturer’s instructions.
5
Quantifying Inflammatory Cytokines in Colonic Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To detect the levels of inflammatory cytokines, the colon tissues of mice were harvested and homogenized in cold PBS buffer containing digestive enzymes, including collagenase I (BioFroxx, cat. no. 1904GR001), collagenase IV (BioFroxx, cat. no. 2091GR001), and Hyaluronidase Grade I (BioFroxx, cat. no. 1141GR001). The concentrations of IL-1β, TNF-α, IL-6, IL-10, and TGF-β in colon tissue were determined by ELISA kits (IL-1β: Invitrogen, cat. no. 88-7013, TNF-α: Invitrogen, cat. no. 88-7324, IL-6: Invitrogen, cat. no. 88-7064, IL-10: Invitrogen, cat. no. 88-7015 and TGF-β: Invitrogen, cat. no. 88-8350) following the manufacturer’s instructions.
6
Tumor Microenvironment Immune Modulation Protocol
7
Isolation of Primary Mouse Lung Fibroblasts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary mouse lung fibroblasts were prepared as follows [18 (link)]: (1) lung tissues obtained from 6–8 weeks old C57BL/6 mice were perfused with 10–20 ml PBS into the right ventricle until the lungs were blood flushed and had a white appearance. (2) The tissue was cut into very small pieces using surgical scissors and then incubated with 0.5 ml of collagenase I (final concentration, 1000 U/ml) (Biofroxx) at 37°C for 30 min. (3) Centrifuge at 1000 g for 5 min, discard the supernatant. (4) Then add 500 μl 0.25% trypsin-EDTA to digest tissues at 37°C for 10 min. (5) After centrifuge, plate the suspension into a 100 mm cell culture dish and adherent cells for 1 hour at 37°C, then discard the supernatant.
The human embryo lung fibroblast cell (HELF) was purchased from iCell Bioscience (Shanghai, China). All cells were cultured in DMEM (11995-065, Gibco) supplemented with 10% fetal bovine serum (FBS, 10099141, Gibco) and 1% penicillin-streptomycin (SV30010, Hyclone) in an atmosphere at 37°C and 5% CO2. Human and mouse IL-19 and TGF-β1 were purchased from GenScript (Nanjing, China). LY2109761 was obtained from Selleck Chemicals (Houston, USA), solubilized in dimethyl sulfoxide (DMSO).
The human embryo lung fibroblast cell (HELF) was purchased from iCell Bioscience (Shanghai, China). All cells were cultured in DMEM (11995-065, Gibco) supplemented with 10% fetal bovine serum (FBS, 10099141, Gibco) and 1% penicillin-streptomycin (SV30010, Hyclone) in an atmosphere at 37°C and 5% CO2. Human and mouse IL-19 and TGF-β1 were purchased from GenScript (Nanjing, China). LY2109761 was obtained from Selleck Chemicals (Houston, USA), solubilized in dimethyl sulfoxide (DMSO).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!