Gelatin (porcine skin, type‐A powder), paraformaldehyde, and streptozotocin (STZ) were purchased from Sigma‐Aldrich (USA). Collagenase type IV, Collagenase type II, and DNase I were purchased from Sangon Biotech (China). DAPI, Red Blood Cell Lysis Solution, and Citrate Antigen Retrieval solution were provided by Beyotime (China). Mouse CD45 MicroBead Kit and Human CD14 MicroBead Kit were purchased from Miltenyi Biotec (Germany). Proteome Profiler Mouse Cytokine Array Kit and Proteome Profiler Mouse Angiogenesis Array Kit were purchased from R&D Systems (USA). RayBio® L‐Series Mouse Antibody Array 308 Membrane Kit was provided by RayBiotech (USA). Corning® Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix was provided by Corning (USA).
Type 2 collagenase
Manufactured by Sangon
Sourced in China
Type II collagenase is an enzyme used in cell isolation and tissue dissociation applications. It specifically cleaves type II collagen, a structural component of cartilage and other connective tissues.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Sangon (2 mentions)
Collagenase type 4, by Sangon (2 mentions)
Red blood cell lysis buffer, by Solarbio (2 mentions)
Red blood cell lysis solution, by Beyotime (1 mentions)
Human cd14 microbead kit, by Miltenyi Biotec (1 mentions)
Matrigel growth factor reduced gfr basement membrane matrix, by Corning (1 mentions)
Proteome profiler mouse cytokine array kit, by R&D Systems (1 mentions)
Proteome profiler mouse angiogenesis array kit, by R&D Systems (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Rbc lysis buffer, by Solarbio (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Percoll, by Solarbio (1 mentions)
70 μm cell strainer, by Jet Biofil (1 mentions)
Collagenase b, by Roche (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
M199 medium, by Thermo Fisher Scientific (1 mentions)
Human cd31 34 45 pe, by BioLegend (1 mentions)
11 protocols using type 2 collagenase
1
Isolation and Characterization of Murine Cells
2
Isolation and Culture of Chicken Preadipocytes
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Chicken preadipocytes from breast muscle tissue were cultured in accordance with the method described by Yuan [21 ], with some modifications. Jinghai Yellow chicken was provided by Jiangsu Jinghai poultry industry group co., LTD. Breast muscle tissue was collected from 14-day-old Jinghai Yellow chicken under sterile conditions. This tissue was washed using phosphate-buffered saline supplemented with penicillin (100 units/ml) and streptomycin (100 μg/ml). The washed tissue was cut into 1 mm3 pieces using surgical scissors and then digested using 2 mg/ml collagenase type II (Sangon Biotech, Shanghai, China) with shaking for 2 h at 37°C. The digested cell suspension was filtrated using 200- and 400-mesh screens and centrifuged at 1500 rpm for 10 min at 22°C to separate the stromal–vascular fraction from undigested tissue debris and mature adipocytes. Stromal–vascular cells were plated onto a 60-mm culture plate at a density of 1×105 cells/ml and cultured with Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12 (DMEM/F12) basic medium [10% (v/v) FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin] in a humidified atmosphere with 5% (v/v) CO2 at 37°C until reaching 90% confluence.
3
Flow Cytometry Analysis of Cells and Wound Tissue
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For flow cytometry analysis in vitro, cells were digested to single‐cell suspension, followed by the incubation with 1% BSA containing the specific fluorescence‐conjugated monoclonal antibodies for 30 min on ice. For the flow cytometry analysis of wound tissue, wounds were excised and cut into pieces on ice and then gently dissociated in lysis buffer (2.5 mg/ml collagenase type II (Sangon Biotech), 2.5 mg/ml collagenase type IV (Sangon Biotech), and 0.5 mg/ml DNase I (Sangon Biotech)) in 1 × HBSS. The resulting cells were passed through a 70 μm cell strainer and suspended in red blood cell lysis buffer. Thereafter, cells were used for specific fluorescence‐conjugated monoclonal antibodies staining, followed by flow cytometry assessment (Attune® NxT Flow Cytometer).
4
Isolation of Bone Tissue Cells
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Bone tissue cells were extracted from the femoral head specimens based on widely used dissociation protocols with a few adjustments [50 (link), 51 (link)]. First, femoral heads were washed three times with αMEM (Cat: SH30265.01, HyClone, USA) and dissected into small pieces of approximately 1-2 mm in diameter. Bone pieces (10 g wet weight) were placed into a 50 ml conical tube with 20 ml of 2 mg/ml collagenase type II (Cat: A004174-0001, Sangon Biotech, China) dissolved in αMEM with 100 U/ml Penicillin and 100μg/ml Streptomycin (Cat: 15140-122, Gibco, USA) and digested with gentle agitation for 25 minutes at 37° C. After that, the collagenase solution was aseptically removed and bone pieces were rinsed in 10 ml PBS for 3 times. Briefly, after five rounds of digestion, we combined the collagenase solutions from the last two rounds of digestion and filtered the solution through a 40 μm filter. Finally, we incubated the collected cells with red blood cell (RBC) lysis buffer (Cat: R1010, Solarbio, China) for 5 minutes and then washed it twice with PBS.
5
Heart-Infiltrating Cell Isolation
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The heart-infiltrating cells were isolated, as previously described (18 (link)). Briefly, the heart tissues were obtained and cut into pieces after which the tissue fragments were digested with 1 mg/mL of collagenase B (Roche 11088815001) or Collagenase Type 2 (Sangon Biotech A004174-0100) in HEPES buffer then rotated gently for 45 min at 37°C. In addition, cell suspensions were filtered with 70-μm cell strainers (BIOFIL) and Percoll (Beijing Solarbio Science & Technology Co., Ltd.) was used to purify heart mononuclear cells through density centrifugation.
6
Isolation and Characterization of Granulosa Cells
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According to the previously published methods the primary culture of GCs from prehierarchical follicles measuring 6.0–8.0 mm in diameter was performed. Briefly, following granulosa layers were immediately isolated from the prehierarchical follicles (6.0–8.0 mm in diameter). After washing with M199 medium (Gibco, New York, NY, USA), granulosa layer cells were dispersed in 0.2% collagenase (type 2; Sangon, Shanghai, China) for 30 min and enhanced with 10% fetal calf serum (Gibco, New York, NY, USA) in humidified chambers at 37 °C, 5% CO2 [2 (link),26 ,27 (link)]. Cultured GC used in experiments were purified and quantified. The specificity of the GC was determined by H & E staining and immunofluorescence [24 (link)].
7
Isolating Human Osteoblasts from Femoral Head
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We isolated osteoblasts from the human femoral head according to a previously widely used protocol with only minor modifications (9 (link)). Briefly, bone tissue was divided into bone fragments with bone forceps and washed with phosphate-buffered saline (PBS). The fragments were placed into the digestive solution containing a highly purified, endotoxin-free type II collagenase (1 mg/ml, Cat: A004174-0001, Sangon Biotech, Shanghai, China) and digested for two rounds at 37℃, the first round for 30 min and the second round for 60 min. The solution was then collected and filtered through a 70 μm filter and incubated with red blood cell lysis buffer for five minutes. The collected cells were washed twice with PBS and incubated with human CD31/34/45-PE (Cat:303106, Cat:343606, Cat:304008, BioLegend, San Diego, CA, USA), and human ALPL-APC (Cat: FAB1448A, R&D Systems, Minneapolis, MN, USA) antibodies. After incubation, ALP+/CD45/34/31− cells were collected as osteoblasts by fluorescence-activated cell sorting (FACS).
8
Primary Human Osteoblast Extraction Procedure
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Human primary
osteoblasts were extracted from femoral heads that were removed during
surgery from patients undergoing femoral head replacement after the
femoral neck fracture. The connective tissue at the end of the femoral
head was dissected in a sterile environment, and then the portion
of the cancellous bone was cut from the sample using a rongeur. The
cancellous bone specimen was completely cut in a Petri dish using
ophthalmic scissors, and after washing, it was digested with type
II collagenase (Sangon, China) to obtain a primary human osteoblast
sample. The cells obtained by the above operation will be inoculated
into a culture flask. When these cells grew to cover 80% of the bottom
of the bottle, the cells were trypsinized and passaged. After induction
of mineralization, alizarin red was used to stain potential mineralization
nodules for the identification of osteoblasts, and then third generation
cells were used for subsequent experiments. Primary human osteoblasts
and MC3T3-E1 cells for subsequent studies were both cultured in an
incubator containing 5% concentration of carbon dioxide using alpha
modification minimum essential medium (α-MEM, HyClone, USA)
containing 10% standard fetal bovine serum (HyClone, USA).
osteoblasts were extracted from femoral heads that were removed during
surgery from patients undergoing femoral head replacement after the
femoral neck fracture. The connective tissue at the end of the femoral
head was dissected in a sterile environment, and then the portion
of the cancellous bone was cut from the sample using a rongeur. The
cancellous bone specimen was completely cut in a Petri dish using
ophthalmic scissors, and after washing, it was digested with type
II collagenase (Sangon, China) to obtain a primary human osteoblast
sample. The cells obtained by the above operation will be inoculated
into a culture flask. When these cells grew to cover 80% of the bottom
of the bottle, the cells were trypsinized and passaged. After induction
of mineralization, alizarin red was used to stain potential mineralization
nodules for the identification of osteoblasts, and then third generation
cells were used for subsequent experiments. Primary human osteoblasts
and MC3T3-E1 cells for subsequent studies were both cultured in an
incubator containing 5% concentration of carbon dioxide using alpha
modification minimum essential medium (α-MEM, HyClone, USA)
containing 10% standard fetal bovine serum (HyClone, USA).
9
Osteoblast Isolation from Human Femur
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Osteoblasts were extracted from the human femur head based on the widely used dissociation protocols [19 (link)] with a few minor adjustments. Briefly, bone tissue samples were chopped into small fragments and washed twice with phosphate-buffered saline (PBS). These fragments, containing a mixture of cortical and trabecular bone, were then incubated with a highly purified, endotoxin-free type II collagenase (1 mg/ml, Cat: A004174-0001, Sangon Biotech, Shanghai, China) at 37.0° C for 30 mins and 60 mins in the first and second round of digestion respectively. After the incubation, the solution was filtered through a 70 μm filter and incubated with red blood cell lysis buffer (Cat: R1010, Solarbio Science and Technology CO., Beijing, China) for 5 mins. The collected cells were washed twice with PBS.
10
Allogenic Tendon-Autologous Cartilage Cells Reconstruction
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To establish the allogenic tendon-autologous cartilage cells reconstruction tool, the cartilage cells were isolated and cultured in vitro (Figure 1B ). The New Zealand rabbits were anaesthetized using 7% chloral hydrate. The articular cartilage was removed under aseptic conditions and washed with PBS. Then, the cartilage cells were extracted by digesting the cartilage chips with type II collagenase (Sangon Biotech. Co., Shanghai, China) at 37°C for 12 h and with 5% CO2 according to a previously reported method [10 (link)]. The suspension of cartilage cells were filtered with a 70-μm nylon mesh, and isolated cartilage cells were washed with PBS solution. The cartilage cells were cultured in DMEM (Gibco BRL, Grand Island, NY, USA), and cartilage cells at passage 3 were used. Cartilage cells were treated with TGF-β1 at final concentrations of 0.1 ng/ml and 0.2 ng/ml for use in further experiments.
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