For the isolation of murine central marrow, the ends of femurs, tibias and iliac crests were removed and the bones flushed using a 21- or 23-gauge needle. For the isolation of endosteal cells, the marrow-depleted bones and the bone ends were ground in a mortar and pestle. The bone fragments were washed, incubated in 3 mg ml−1 Collagenase I (Roche, Basel, Switzerland) and 4 mg ml−1 Dispase II (Worthington, Lakewood, NJ), in phosphate-buffered saline (PBS; 310 mOsm) in a shaking incubator (37 °C; 250 r.p.m. for 5 min), then washed by vigorously shaking66 (link). RBC in collected PB were lysed using NH4Cl lysis buffer for 5 min at room temperature.
Dispase 2
Manufactured by Worthington
Sourced in United States
Dispase II is a neutral protease enzyme isolated from Bacillus polymyxa. It is commonly used for the dissociation of a variety of cell types, including epithelial, endothelial, and fibroblast cells, from their extracellular matrix.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase 1, by Worthington (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Worthington (2 mentions)
Collagenase 1, by Roche (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Serva Electrophoresis (1 mentions)
Gentamicin, by Serva Electrophoresis (1 mentions)
Recombinant human insulin, by Merck Group (1 mentions)
Collagenase type 1, by Serva Electrophoresis (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Facscalibur, by BD (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
N2 supplement, by Thermo Fisher Scientific (1 mentions)
Basic fgf, by Merck Group (1 mentions)
Percoll, by Merck Group (1 mentions)
Papain, by Worthington (1 mentions)
Efluor 780, by Thermo Fisher Scientific (1 mentions)
Efluor 450, by Thermo Fisher Scientific (1 mentions)
10 protocols using dispase 2
1
Isolation of Murine Endosteal Cells
2
Single-cell Isolation from Tissue Samples
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Tissue samples were minced to 1–2 mm pieces and enzymatically digested in DMEM/F12 (Gibco, TFS) containing 2% bovine serum albumin (BSA; Serva, Heidelberg, Germany), 5 mg·mL−1 recombinant human insulin (Sigma‐Aldrich), 0.5 mg·mL−1 hydrocortisone (Sigma‐Aldrich), 50 mg·mL−1 gentamicin (Serva), 2 mg·mL−1 collagenase type I (cat. no. LS 004194; Worthington, Lakewood, NJ, USA), 0.6 U·mL−1 dispase II (cat. no. 04942078001; Roche, Basel, Switzerland) and 10 mm Y‐27632 dihydrochloride (ROCK inhibitor, Santa Cruz Biotechnology, Dallas, TX, USA), for 14 h at 37 °C using 10 rpm agitation. Samples were then treated with 15 mg·mL−1 DNase I (cat. no. 04942078001; Roche) for 5 min at 37 °C, washed with PBS, and filtered through a 70 μm strainer. Red blood cells were lysed with ACK buffer (155 mm ammonium chloride, 10 mm potassium bicarbonate, and 100 μm EDTA solution in sterile MQ water) at 37 °C for 5 min, washed with PBS and incubated with CD24 and ROR1 antibodies 30 min at room temperature. After washing once with CSB buffer, cells were stained with 1 μm cisplatin for subsequent dead cell exclusion (Fluidigm) and quenched with CSB buffer. Cells were then fixed with 1.6% paraformaldehyde (TFS) for 15 min at room temperature and stored at −80 °C in 10% glycerol in fetal bovine serum.
3
Isolation of Rat DRG and Spinal Cord Progenitor Cells
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All animal experiments were approved by institutional animal care and use committees. Male and female Sprague Dawley rats (weight, 150-225 g) were used for the culturing of DRGs. No differential responses were noted in DRGs harvested from male versus female animals in the studies below. For injury conditioning, rats were subjected to sciatic nerve crush at mid-thigh, as previously described (Twiss et al., 2000 (link)). Seven days after nerve crush, animals were killed and L4/5 DRGs were isolated for dissociated culture preparations.
Progenitor cells were isolated from spinal cords of rat embryos (E13.5) as previously described (Bonner et al., 2011 (link)). Briefly, embryos were placed in DMEM/F-12 and the meninges removed by incubation in collagenase I (10 mg/ml; Worthington) plus dispase II (20 ng/ml; Worthington) in HBSS for 9 min at room temperature. Spinal cords then were then treated with trypsin (0.5%)/EDTA for 20 min at 37°C, dissociated, and plated as outlined below.
Progenitor cells were isolated from spinal cords of rat embryos (E13.5) as previously described (Bonner et al., 2011 (link)). Briefly, embryos were placed in DMEM/F-12 and the meninges removed by incubation in collagenase I (10 mg/ml; Worthington) plus dispase II (20 ng/ml; Worthington) in HBSS for 9 min at room temperature. Spinal cords then were then treated with trypsin (0.5%)/EDTA for 20 min at 37°C, dissociated, and plated as outlined below.
4
Separation of Tooth Germ Tissues for RNA Analysis
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Incisor and molar tooth germs were dissected from P0 and P10 mice using a dissection microscope. To separate epithelium and mesenchyme, the tooth germs were treated with dispase II and collagenase I (Worthington) for 30 min at 37 °C. This procedure separates the epithelium from the mesenchyme and allows for specific RNA extraction of the two tissue types (31 (link)). Total RNAs including miR were prepared using miRNeasy Mini Kit from Qiagen. LC Sciences performed the miR microarray analyses.
5
Isolation and Analysis of Primary Cardiac Cells
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Primary cardiac cells were prepared from WT and Tg mice according to previous protocol with modification [10 (link)]. Briefly, cardiac tissue from WT and Tg mice was digested with 0.1% DispaseII (Worthington, Lakewood, USA) at 4°C over night and then 0.125% Trypsin (Gibco, Grand Island, NY, USA) at 37°C for 10 minutes. The primary cardiac cells were cultured in DMEM (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) at 37°C for 24 hours. After treatment with LPS, the primary cardiac cells were stained with both propidiumiodide (PI) and annexin V (AV)-FITC and loaded on a flow cytometer (FACSCalibur, Becton Dickinson, Franklin Lakes, USA). Data from 10,000 cells/sample were collected, and the quadrants were set according to the population of viable, unstained cells in untreated samples.
6
Isolation and Culture of Neural Progenitor Cells from Mouse Hippocampus
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NPC cultures derived from adult mice hippocampus were performed as previously described25 (link). Briefly, mice were deeply anesthetized using halothane and decapitated, and brains were removed. Hippocampi were dissected, pooled, and dissociated in DMEM (Invitrogen, CA) containing 2.5 U/ml papain, 1 U/ml dispase II, and 250 U/ml DNase 1 (Worthington Biochemical, NJ) for 45 min at +37°C. Dissociated cells were purified on two Percoll (Sigma, MI) gradients, 35 and 65%. The resulting cells were plated at 1 × 104 cells per well in uncoated 12-well culture plates in DMEM/F-12, supplemented with 15 mm HEPES, 2 mm l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml Amphotericin B (Calbiochem, CA), N2 supplement (Invitrogen, CA), and 20 ng/ml basic FGF (Sigma, MI).
7
Skin Leukocyte Isolation and Analysis
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Three 6 mm punch biopsies from each animal were performed and tissues pooled for processing and staining. Skin specimens were digested using 2 mg ml−1 Dispase II, 2 mg ml−1 Collagenase Type IV (Worthington Biochemical, Lakewook, NJ), 200 U ml−1 DNase I (Worthington Biochemical, Lakewook NJ), 2 mM Ca2+Cl2 and 2 mM Mg2+Cl2 for 45 min at 37 °C. Single-cell suspensions were counted in a Neubauer cell chamber and stained with the following antibodies (all from Biolegend, San Diego, CA): CD45-PerCP/Cy5.5 (clone 30-F11), CD11b-APC/Cy7 (clone M1/70), Ly6G-FITC (clone A1-8) and Siglec F-PE (clone E50-2440). Nonspecific staining was prevented with CD16/CD32 blockade (clone 24G2). Dead cells were excluded using the fixable viability dye eFluor780 or eFluor450 (eBiosciences, San Diego, CA). Flow analysis was performed on an LSR-II Flow Cytometer and analysed using Flow Jo Software. Total number of cellular infiltrates was calculated from relative frequencies.
8
Isolation and Culture of Human Skin Cells
9
Isolation and Culture of Dental Pulp Cells
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Dental pulp cells (DPCs) were isolated from the extracted incisors of 8-week-old female Sprague-Dawley rats following an established protocol with modification [31] (link). Briefly, the gingival soft tissues were excised and incisors were then extracted using forceps. The incisors were washed with phosphate-buffered saline (PBS) supplemented with 2% penicillin/streptomycin (PS; Thermofisher Scientific). After removing the soft tissues from the root surface, the incisors were split open and the pulp tissues were carefully extracted. These pulp tissues were cut into small pieces and digested in 2% dispase II and 0.2% collagenase I (Worthington, Lakewood, NJ, USA) at 37°C for 1 h. The cell suspension was passed through a 40-μm cell strainer (Corning®, Corning, NY, USA) to disperse into single cells before seeding at a density of 2 × 104 cells/cm2. The cells were grown in complete culture medium composed of DMEM-F12 supplemented with 10% FBS and 1% PS with medium change every alternate day. Upon confluence, DPCs were dissociated with TrypLe™ (Thermofisher Scientific) and sub-cultured to passage (P) 3 for subsequent experiments.
10
Enzymatic Muscle Biopsy Dissociation
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Muscle was transported from the operation suite to the laboratory within 15 min in ice-cold wash-buffer. Upon arrival the muscle biopsy was initially dissected free of visible tendon/connective tissue and fat. The biopsy was then divided into pieces of up to 0.5-0.8 grams and briefly mechanically minced with sterile scissors. The muscle slurry was then transferred to C-tubes (cat nb. 130-093-237, Miltenyi Biotec, Lund, Sweden) containing 8ml wash buffer including 700 U/ml Collagenase II (lot 46D16552, Worthington, Lakewood, NJ, USA) and 3.27 U/ml Dispase II (cat nb. 04 942 078 001, Roche Diagnostics, Basel, Switzerland). Mechanical and enzymatic muscle digestion was then performed at 37°C on the gentleMACS with heaters (cat nb. 130-096-427, Miltenyi Biotec) for 60 min using a skeletal muscle digestion program (37C_mr_SMDK1). When digestion was complete 8 ml wash buffer was added to the single cell solution and this was filtered through a 70µm cell strainer and washed twice to collect any remaining cells. The suspension was centrifuged at 500g for 5 min and the supernatant removed. The cell pellet was resuspended in freezing buffer (StemMACS, cat nb. 130-109-558, Miltenyi Biotec) and stored 1-3 weeks at -80.
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