Established A549 and H1299 cell lines were purchased from Fuheng Biotechnology (Shanghai, China). All the cell lines were validated by short tandem repeat analysis. Patient-derived primary LUAD cells were established from LUAD tissues as previously described [49 (link)]. Briefly, fresh tissues less than 1.0 cm3 without necrosis were immediately rinsed 3 times with cold Dulbecco’s Phosphate Buffered saline and then re-suspended in Dulbecco’s Modified Eagle Medium (DMEM) containing collagenase I (2 mg/ mL, Solarbio, Shanghai) at 37 °C for 4 h. After being rinsed with DMEM 3 times, cells were cultured at 37 °C, 5% CO2 condition. For monolayer culture, cells were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin. For 3D spheroid culture, basement membrane extract (BME) (Trevigen, Gaithersburg, MD, USA) was seeded in a 96-well plate at 50 μl/well and pre-warmed at 37 °C for 0.5 h. Then, cells were seeded on top of the plate coated with BME at a density of 105 cells per well. Images were captured using a microscope [49 (link)].
Collagenase 1
Manufactured by Solarbio
Sourced in China
Collagenase I is an enzyme used to dissociate and isolate cells from connective tissue. It breaks down collagen, a major structural component of the extracellular matrix.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Basement membrane extract, by Bio-Techne (1 mentions)
Mem non essential amino acid solution, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
C8140, by Solarbio (1 mentions)
Niacin, by Merck Group (1 mentions)
Butyrate, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Wisent (1 mentions)
Epidermal growth factor (egf), by Corning (1 mentions)
Penicillin, by Solarbio (1 mentions)
Streptomycin, by Solarbio (1 mentions)
Dimethyl sulfoxide (dmso), by Solarbio (1 mentions)
T aoc, by Nanjing Jiancheng (1 mentions)
Phosphate buffered saline (pbs), by Solarbio (1 mentions)
Amphotericin b, by Solarbio (1 mentions)
Dnase 1, by Solarbio (1 mentions)
Penicillin streptomycin mixing liquid, by Solarbio (1 mentions)
Foxp3 pe, by Thermo Fisher Scientific (1 mentions)
23 protocols using collagenase 1
1
Establishment of Lung Cancer Cell Lines
2
Isolation and Differentiation of Mouse Adipose-Derived Stromal Cells
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Stromal-vascular fractions (SVF) were isolated as previously described [5 (link),12 (link)]. Briefly, inguinal white adipose tissues were dissected from 8-week-old mice, minced and digested in 1.5 %BSA KRBH containing 1 mg/ml collagenase I (Solarbio, China) at 37 °C for 1h. Digested tissue was filtered through 100 μm nylon mesh and centrifuged to separate floating adipocytes. The resulting pellet (SVF) was cultured in DMEM, supplemented with 10 % FBS, 1 % MEM non-essential Amino acid solution (11140050, Gibco), 1 % GlutaMax (35050061, Gibco) and 100U/ml penicillin and streptomycin (growth medium). Adipogenesis was induced by incubation in growth medium supplemented with 1x ITS (Gibco), 1 μM dexamethasone, 33 μM Biotin, 2 nM T3, 17 μM Pantothenate, 0.5 mM IBMX and 1 μM rosiglitazone (differentiation medium). After 2–4 days, the medium was changed to differentiation medium without IBMX and rosiglitazone (maintenance medium), and then changed again every 2 days until full differentiation (i.e., day 8–10). For activin B treatments, matured adipocytes were serum starved for 2h and then incubated with indicated concentration of activin B for either 20min for detection of SMAD phosphorylation levels, or 24h for RNA extraction. For CL-316,243 treatment, matured adipocytes were incubated with 10 μM CL-316,243 for 20min before harvested for protein extraction.
3
Rat Alveolar Type II Cell Culture
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As previously described (Hou et al. 2015 (link)), lung tissues were extracted, rinsed repeatedly and then digested with 0.25% trypsin EDTA (25200056, GIBCO, USA) for 30 min followed by with 0.1% collagenase I (C8140, Solarbio, USA). The rat alveolar type II epithelial cell line RLE-6TN (Mount Sinai School of Medicine, New York, NY) cells were randomly divided into a model group ((85% O2; 5% CO2) and a control group (21% O2; 5% CO2) and harvested after 12, 24, and 48 h of culture for the subsequent experiments. The siRNA sequence that targets rat Rad1 (siRad1) was designed and synthesized by Shanghai Hanbio Biotechnology Co. Ltd. (Shanghai, China) and transfected with Lipofectamine 2000 (Thermo Fisher Scientific, USA). The plasmid DNA clones for rat Rad1 and the empty vector were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China).
4
Butyrate and Niacin's Impact on Cell Viability
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Butyrate and niacin were obtained from Sigma, and Dulbecco's modified Eagle's medium (DMEM) was from Wisent. Phosphate-buffered saline (PBS), MTT, dimethyl sulfoxide (DMSO), amphotericin B, penicillin and streptomycin, 0.25% trypsin +0.02% EDTA, and 0.1% collagenase I were purchased from Solarbio. Epidermal growth factor (EGF) was obtained from Corning. ITS was purchased from ScienCell. Fetal bovine serum (FBS) was from BI, and ROS, T-AOC, and Annexin V-FITC/PI kits were obtained from Jiancheng.
5
Isolation and Characterization of Murine Lung Fibroblasts
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Newborn 3–7 days C57BL/6 mice were killed, and the lungs from the thoraxes were obtained under sterile conditions. The lung was washed twice with sterile PBS containing 20% concentration of penicillin streptomycin mixing liquid (Solarbio, China). Subsequently, the lung tissue was chopped with surgical scissors until finely minced, digested with 2 ml enzyme mix (1 mg/ml Collagenase I and 10 μg/ml DNase I (Solarbio, China), and incubated at 37 °C for 1 h with gentle agitation. Further, an 80 μm cell strainer was used to filter the cells. Following, the cells were resuspended in Dulbecco’s modified Eagle’s media and Ham’s F-12 (DMEM/F-12) (Hyclone, USA) containing 10% fetal calf serum (FBS) (CellMax, Australia), 1% penicillin streptomycin mixing liquid (Solarbio, China) and incubated at 37 °C in a humidified atmosphere of 5% CO2. We performed anti-Vimentin immunofluorescence staining to identify cells for >90% fibroblasts, and performed anti-Prosurfactant protein C (proSP-C) immunofluorescence staining to identify cells for <5% alveolar epithelial cells. Cells were used between the third and sixth passages and photographed.
6
Melanoma-Specific Peptide Protocol
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Chitosan (Mw 7 kDa, deacetylation degree > 95%) was purchased from Aoxing Bio-Technology Co. (Zhejiang, China). Folate was purchased from Bio Basic Inc. (Markham, ON, Canada). Monoclonal antibodies against mouse CD11b-FITC and Ly6G-PE were obtained from Beckman Coulter (Brea, CA, USA). Monoclonal antibodies against mouse CD4-FITC, CD8-PerCP-Cy5.5, CD25-PE-Cy5, CXCR3-FITC, Foxp3-PE and Foxp3 staining buffer were purchased from eBiosciences (San Diego, CA, USA). The anti-CD31 monoclonal antibody was obtained from Abcam (Cambridge, MA, USA). Purified hamster anti-mouse CD28 antibody was purchased from BD Biosciences (San Jose, CA, USA). The anti-mouse PCNA monoclonal antibody was supplied by Boster Bio-Technology Co. (Wuhan, China). TdT-mediated dUTP-biotin nick end labeling (TUNEL) cell-death fluorescein detection kit was obtained from Roche Diagnostics (Hoffmann-La Roche, Basel, Switzerland). Dynal mouse CD8+ T isolation kit was purchased from Invitrogen (Carlsbad, CA, USA). Collagenase I was acquired from Solarbio Co. (Beijing, China). The H-2Kb-restricted, melanoma-specific antigenic peptide TRP2 (amino acids 180 - 188, N-SVYDFFVWL-COOH) was chemically synthesized by Nanjing GenScript Corporation (Nanjing, China) with a purity > 95%.
7
Isolation and Culture of Bovine Adipose-Derived Cells
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Abdominal subcutaneous fat was taken from three Xianan fetal cows of about three months’ size, and the adipose tissue was washed twice with phosphate buffer solution (PBS) containing 2% Penicillin-Streptomycin Solution (Procell, Wuhan, China), and then cut into a mud with scissors. Afterwards, the tissue was added to a digestion solution containing 0.2% Collagenase I (Solarbio, Beijing, China) and placed in a 37 °C water bath to digest the tissue into a flocculent mass.
After digestion, the cells were filtered through a 40 mesh sieve, a 200 mesh sieve, and a 400 mesh sieve in order to remove undigested tissues. After centrifugation at 1000 rpm for 5 min, the cells were resuspended in DMEM/F12 medium containing 10% fetal bovine serum (FBS) and %1 Penicillin-Streptomycin Solution and inoculated at a density of approximately 1 × 106 cells into 25 cm2 culture flasks. After adding complete medium (DMEM/F12 containing 10% FBS and 1% Penicillin-Streptomycin Solution) to the culture flasks, the flasks were incubated at 37° in an incubator containing 5% CO2.
After digestion, the cells were filtered through a 40 mesh sieve, a 200 mesh sieve, and a 400 mesh sieve in order to remove undigested tissues. After centrifugation at 1000 rpm for 5 min, the cells were resuspended in DMEM/F12 medium containing 10% fetal bovine serum (FBS) and %1 Penicillin-Streptomycin Solution and inoculated at a density of approximately 1 × 106 cells into 25 cm2 culture flasks. After adding complete medium (DMEM/F12 containing 10% FBS and 1% Penicillin-Streptomycin Solution) to the culture flasks, the flasks were incubated at 37° in an incubator containing 5% CO2.
8
Isolation and Differentiation of Bovine Muscle Cells
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The longest dorsal muscle of a three-month-old calf was taken and isolated into muscle cells. Skin, fascia, and other tissues were stripped from the longest dorsal muscle tissue of fetal bulls, and the muscle tissue was minced into the smallest possible pieces with scissors. The tissue was shaken with collagenase I (Solarbio, Beijing, China) for 1 h at 37 °C and then centrifuged at 1500× g to obtain the precipitate. Subsequently, trypsin was added for 20–25 min, and digestion was terminated with a DMEM medium containing 10% fetal bovine serum (FBS) (Gibco, Waltham, MA, USA). The cells were then filtered through 100 mesh and 70 mesh filters and centrifuged at 1500 rpm to obtain a cell precipitate. Finally, the cells were resuspended in a medium containing 20% FBS (Gibco, Waltham, MA, USA) and 1% penicillin-streptomycin (Gibco, Waltham, MA, USA). After 2 h of incubation in a 37 °C incubator, the cell cultures and supernatants were inoculated into cell culture plates at 37 °C and 5% CO2 for a subsequent passaging culture. When cell proliferation reached 80%, a differentiation medium containing 2% horse serum (Gibco, Waltham, MA, USA) was used instead of the growth medium to induce cell differentiation.
9
Isolation and Characterization of Mandibular Bone Marrow Mesenchymal Cells
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ABMMCs were isolated and cultured using previously described methods [26 (link)]. Mandibles without molars or soft tissue were obtained from 4-week-old Wistar rats, and then digested with 3 mg/mL collagenase I (Solarbio) and 4 mg/mL dispase II (Roche) at 37 °C for 1 h. Cells were then collected and cultured in primary medium containing α-minimal essential medium (α-MEM; BasalMedia), 15% fetal bovine serum (FBS; Lonsa Science Srl), and 10,000 U/mL penicillin-streptomycin (Biosharp). The ABMMCs used in this study were 3–5 generations.
Flow cytometry was used to identify the phenotype of ABMMCs. In brief, ABMMCs were resuspended with phosphate-buffered saline (PBS; BasalMedia). Then, CD44, CD31 (BD; Biosciences), CD90, and CD45 (Elabscience) antibodies were applied to detect the phenotype of ABMMCs as directed by the manufacturer.
Flow cytometry was used to identify the phenotype of ABMMCs. In brief, ABMMCs were resuspended with phosphate-buffered saline (PBS; BasalMedia). Then, CD44, CD31 (BD; Biosciences), CD90, and CD45 (Elabscience) antibodies were applied to detect the phenotype of ABMMCs as directed by the manufacturer.
10
Neuronal Cell Viability Assay
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N-methyl-D-aspartic acid (NMDA, Shanghai Yuanye Biology company); DMEM medium (Sigma-Aldrich company); trypsin (Amresco company); D-Hank’s solution (Sigma-Aldrich company); cytarabine (Ara-C, Sigma-Aldrich company); CCK-8 Kit (Shanghai Bogu Biotechnology Co., Ltd.); malondialdehyde kit (Nanjing Jiancheng Bioengineering Research Institute); superoxide dismutase kit (SOD, Nanjing Jiancheng Biology); GSH-PH Kit (Nanjing Jiancheng Bioengineering Institute); DMEM/F-12 medium (Hyclone company); PBS solution (1x, Solarbio company); trypan blue (Solarbio company); collagenase I (Solarbio company); cytarabine (Solarbio company); L-glutamine (Solarbio company).
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