Adult mouse dorsal root ganglia (DRG) neuron culture was performed as previously described (33 (link)). Briefly, the ganglia were dissociated with 100 U of papain (P4762, Sigma) followed by 1 mg/ml collagenase-II (11179179001, Roche) and 1.2 mg/ml dispase-II (04942078001, Roche). The DRGs were triturated in 1xHBSS, 10 mM glucose, and 5 mM Hepes (pH 7.35) using a fire-coated Pasteur pipette and then layered on 20% percoll in L15 media and recovered by centrifugation at 1000g for 8 min. Cells were washed briefly in growth media (F12, 10% FBS, Primocin (100 μg/ml, InvivoGen #ant-pm-1)) and plated on poly-L-lysine (P4832, Sigma) and laminin (23017-015, Invitrogen)-coated glass coverslips. Culture media and serum were purchased from Thermo Fisher Scientific. DRG neurons were grown for 24 to 48 h in culture at 37 °C and 5% CO2.
Collagenase 2
Manufactured by Roche
Sourced in United States
Collagenase II is a lab equipment product used for the enzymatic dissociation of various tissue types. It is a mixture of proteolytic enzymes that specifically target and cleave collagen, a key structural component in the extracellular matrix. This product facilitates the isolation and recovery of cells from solid tissues.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Merck Group (7 mentions)
Dispase 2, by Roche (6 mentions)
Dispase, by Worthington (5 mentions)
Dnase 1, by Roche (3 mentions)
Collagenase, by Roche (3 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
Laminin, by Thermo Fisher Scientific (2 mentions)
P4762, by Merck Group (2 mentions)
Ant pm 1, by Merck Group (2 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Percoll rpmi gradient, by GE Healthcare (2 mentions)
Facsaria cell sorter, by BD (2 mentions)
Dmem f12, by Roche (2 mentions)
Primocin, by InvivoGen (1 mentions)
Collagenase 4, by Merck Group (1 mentions)
Dba 1lacj mice, by Jackson ImmunoResearch (1 mentions)
Chick type 2 collagen, by Chondrex (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
27 protocols using collagenase 2
1
Mouse Dorsal Root Ganglia Neuron Culture
2
Collagen-Induced Arthritis Model in Mice
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Mice (female DBA/1Lacj mice, Jackson Laboratories; 8–10 weeks) were housed with 12 hours light-dark cycle with food and water provided ad libitum. Following acclimation, mice were immunized with an emulsion containing equal amounts of Chick Type II Collagen (Chondrex, Redmond, WA) and Complete-Freund’s adjuvant (CFA, Chondrex), 100 μg each per mouse, on day 0 at the base of the tail and administered a second immunization boost on day 21. On day 21, animals were examined for clinical arthritis scores and randomized into treatment groups. JNJ-54271074 (0.3, 3, 10, 30, or 60 mg/kg) or vehicle (20% HPCD) were given orally, twice-daily (8 hour apart) from day 21 to 35. The clinical arthritis scores were evaluated from days 21 to 34. Animals were euthanized under CO2 on day 35. Both hind-paws from each animal were collected and fixed in 10% neutral buffered formalin for histopathology. Both front paws were digested after removing the skin in 0.125% (v/v) Dispase II (Roche, Indianapolis, IN), 0.2% collagenase II (Roche, Indianapolis, IN), and 0.2% collagenase IV (Sigma-Aldrich, St. Louis, MO) in HBSS for 75 min at 37 °C and passed through a cell strainer. Cells were then stimulated and analyzed by flow cytometry for cytokine-producing cells.
3
Isolation and Expression Analysis of Lymphatic Endothelial Cells
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Ear skin was dissected and minced in 5 mg/ml Collagenase II (Roche) and 0.2 mg/ml DNaseI (Roche) digestion mix. From FACS-sorted LECs (CD31+/PDPN+) total RNA was extracted by RNeasy Micro kit (QIAGEN) and all obtained RNA was reverse transcribed using oligo dT (SuperScript III First-Strand Synthesis System, Invitrogen). cDNA was pre-amplified using the TaqMan PreAmp Master Mix Kit. Gene expression levels were analysed using TaqMan Gene Expression Assay (AppliedBiosystems) and the StepOne Plus Real-Time PCR system (Applied Biosystems) following manufacturer’s instructions. Relative gene expression levels were normalized to GAPDH. The following probes were used: Gapdh Mm_03302249_g1, Efnb2 Mm00438670_m1 (ThermoFisher Scientific).
4
Isolation of Intestinal Leukocytes
5
Intestinal Leukocyte Isolation and Analysis
6
Isolation and Culture of Myocardial Cells
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Animal experiments have been approved by the Animals Welfare Committee of Wenzhou Medical University. All the animals received human care. H9C2 cells were obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). Cells were grown as a monolayer culture in high glucose DMEM (Gibco, Carlsbad, CA, USA) supplemented with 10% foetal bovine serum (FBS; Gibco). Mouse myocardial cells were prepared from neonatal (born within 3 days) C57/B6J mice. Heart tissues were repeatedly digested by collagenase II (Roche, Basel, Switzerland) in 37°C water bath. All myocytes were filtrated in a 200 mesh strainer. Cells remained in suspension were seeded in 6‐well plates at a density of 1 × 105 cells per well with a differential plating step of 1 hr 19 . The medium used was consisted of high glucose DMEM, 20% FBS, 1% penicillin and 1% streptomycin. The cultures were maintained in a humidified incubator with 5% CO2 at 37°C.
7
Isolation of Intestinal Leukocytes
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Intestinal tissues were sequentially treated with PBS containing 1 mM DTT at room temperature for 10 min, and 5 mM EDTA at 37°C for 20 min to remove epithelial cells, and then minced and dissociated in RPMI containing collagenase (1 mg/ml collagenase II; Roche), DNase I (100 μg/ml; Sigma), dispase (0.05 U/ml; Worthington) and 10% FBS with constant stirring at 37°C for 45 min (SI) or 60 min (LI). Leukocytes were collected at the interface of a 40%/80% Percoll gradient (GE Healthcare). The Peyer’s patches and cecal patch were treated in a similar fashion except for the first step of removal of epithelial cells. Lymph nodes and spleens were mechanically disrupted.
8
Isolation of Immune Cells from Tissues
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For isolation of LP cells, single‐cell suspensions were generated as previously described.15 In some instances (see Section 3 ), Liberase TM (0.3 WuenschU/mL, Roche) was replaced with Collagenase II (230 U/mL, Gibco). For isolation of cDC from non‐digested intestine and lung, tissues were cut into small pieces, placed on a petri‐dish filled with R10 medium (RPMI 1640 containing with 10% FCS (Sigma‐Aldrich), 1 mM sodium pyruvate (Gibco), 10 mM HEPES, 100 U/mL penicillin and 100 μg/mL streptomycin, 50 μg/mL gentamycin (Gibco), 50 μM 2‐mercaptoethanol (Gibco)) at 37°C, 5% CO2, and cells collected from the culture after 20 hours. For isolation of MLN and splenic cells, organs were mashed through a 70 μm cell strainer, except in Figure 3D where MLN were digested with Collagenase II (230 U/mL) and DNAse I (30 μg/mL, Roche), as described previously.15 Red blood cells in splenic cell suspensions were lysed using ACK lysing buffer.
9
Quantifying Transplanted Cardiac Cell Survival
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To quantify the survival rate of post-transplanted CDCs, heart cells were isolated from the mouse heart at different time points after CDC transplantation using a method of enzymatic digestion [6 (link)]. Briefly, heparinized animals were anesthetized and the hearts were rapidly mounted to a langendorff apparatus. The hearts were perfused retrogradely with Ca2+-free normal Tyrode’s solution containing 1 mg/mL collagenase II (Roche) and 0.1 mg/mL protease (Sigma) for about 10 min, depending on digesting conditions. The ventricles were cut off and minced in solution, followed by pipetting to disassociate the cells, and then filtered through a nylon mesh to remove big pieces of undigested tissues. The GFP+ cells represented CDCs or their derived cells, which were carefully counted. The survival rate = 100% × (number of survived GFP+ cells/1 × 105).
10
Isolation and Culture of Muscle Stem Cells
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Isolation and culture of MuSCs were performed as previously described (Hayhurst et al, 2012 (link)). Briefly, skeletal muscle tissue was isolated from the TA and GA of neonatal mice. The muscle tissue was shredded by sterilized scissors, incubated with 0.2% (wt/vol) collagenase II (Roche) in DMEM containing 20% FCS and antibiotic–antimycotic (100×) (Gibco) for 30 min at 37°C, and dissociated into single myofibers by pipetting several times. The dissociated tissue was then centrifuged at 4,400g for 15 s and suspended with 20% FCS DMEM. The cell suspension including MuSCs was seeded onto a six-well plate coated with Matrigel. The attached cells including MuSCs were then expanded in 20% FCS DMEM condition for a few days. After reaching confluence, the MuSCs were cultured to induce myotubes in DMEM containing 2% HS for 3 d.
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