Preparation of limb muscle for flow cytometric analysis was adapted from Liu et al. [28 (link)]. In short, dissected tissue was minced thoroughly to small pieces in F10 medium (Lonza) containing collagenase II (750 U/ml; Fisher Scientific) using scalpels. Minced tissue was dissociated for 70 min in F10 medium containing 750 U/ml collagenase II), then for 30 min in F10 medium containing collagenase II (100 U/ml) and dispase (1.1 U/ml; Fisher Scientific). Cell suspensions were filtered over a 40 μM cell strainer (Falcon) and a small sample was retrieved to determine total mononuclear cell count using a hematocytometer. The cell suspensions were stained with CD31-APC (1:100), CD45-APC (1:100), Sca1-FITC (1:100) and Vcam-biotin (1:50) primary antibodies. Vcam was visualized using streptavidin-PECY7 (1:100). All antibodies for flowcytometry were derived from BD Biosciences. Cell viability was determined by staining with 1 μg/ml Hoechst 33258 (Sigma). Samples were analyzed on a BD-ARIAIII (BD biosciences).
Collagenase 2
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, United Kingdom, China
Collagenase II is a laboratory enzyme used for the dissociation and isolation of cells from tissue samples. It is a proteolytic enzyme that specifically cleaves the collagen found in the extracellular matrix.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (64 mentions)
Dispase, by Thermo Fisher Scientific (42 mentions)
Trypsin, by Thermo Fisher Scientific (33 mentions)
Dnase 1, by Merck Group (32 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (26 mentions)
Dnase 1, by Thermo Fisher Scientific (24 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (22 mentions)
Percoll, by GE Healthcare (15 mentions)
Trypsin edta, by Thermo Fisher Scientific (15 mentions)
Penicillin, by Thermo Fisher Scientific (13 mentions)
Collagenase 4, by Thermo Fisher Scientific (13 mentions)
Dispase 2, by Thermo Fisher Scientific (13 mentions)
Dispase 2, by Merck Group (12 mentions)
Bovine serum albumin (bsa), by Merck Group (12 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (11 mentions)
Streptomycin, by Thermo Fisher Scientific (11 mentions)
Dmem f12, by Thermo Fisher Scientific (11 mentions)
Tryple, by Thermo Fisher Scientific (11 mentions)
Dnase 1, by Roche (10 mentions)
Horse serum, by Thermo Fisher Scientific (9 mentions)
403 protocols using collagenase 2
1
Dissociation and Immunophenotyping of Limb Muscle
2
Zebrafish Tumor Cell Viability Assay
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Tumors from Tg (dβh-MYCN:dβh -eGFP; dβh-DDX1:CryAA-mCerulean) double transgenic fish and Tg (dβh-MYCN:dβh-eGFP) were excised from adult fish immediately after hypothermal shock euthanasia. Tumors were dissociated using a Collagenase II (Thermo Fisher)–based protocol (330 μL Collagenase II—final concentration: 100 U/mL, 120 μL HBSS, 50 μL FCS for 30 minutes at 37°C, followed by 5 minutes of incubation at 37°C after the addition of 200 μL of Dispase II [Thermo Fisher]—final concentration: 2 U/mL, gently pipetting every 10 minutes). After dissociation, single-cell suspension was transferred to Round-Bottom Polystyrene Test Tubes with Cell Strainer Snap Cap to remove undissociated tissue. Cells were resuspended in DMEM (Gibco) supplemented with 10% FCS and 1% penicillin/streptomycin and plated in 96-well plates at a density of 0.1 × 105 cells. Cells were incubated at 27°C, 5% CO2. After 24 hours, cells were visually inspected for eGFP expression using an AXIO microscope (Zeiss), and rapamycin (2.5 μmol/L) or vehicle was added to the DMSO; cells were then incubated for 72 hours, and viability was assessed through MTT assay (Abcam) following the manufacturer's protocol. Absorbance was measured using an Epoch plate reader (BioTeK) at OD = 590.
3
Tissue Dissociation and Cell Spreading
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Fixed ~5–7 mm3 tissue/tumor solid tissues were enzymaticaly dissociated in collagenase II (10 U/μl) (Life Technologies) and spread on microscopic slides as cells monolayers.1 (link) Briefly, samples were cut into ~2 mm3 pieces, placed in 2 ml tubes filled with collagenase II, 37 °C, and digested for 2 h (tumor tissues) or 45 min (fetal tissues). The resulting digested tissue pieces were rinsed in distilled water, placed in 45% acetic acid for 15 min (maceration) and then applied to slides for spreading. Slides were frozen on dry ice, coverslips removed, and dried at room temperature.1 (link),3 (link)
HT-29 cells were fixed directly in T-flasks or on microscope slides. HT-29 cells were also observed live using phase contrast optics and/or fluorescence microscopy.
HT-29 cells were fixed directly in T-flasks or on microscope slides. HT-29 cells were also observed live using phase contrast optics and/or fluorescence microscopy.
4
Retrograde Labeling of Colon Neurons
5
Isolation and Culture of Muscle Stem Cells
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To obtain purified SCs, primary cells were isolated as described previously (Paris et al., 2016 (link)). Muscles were dissected from mice and digested using collagenase II (Gibco, Carlsbad, California) in Dulbecco's modified Eagles medium (DMEM, Sigma-Aldrich) for 60 min at 37°C with agitation. The suspension was then washed and digested in collagenase II and dispase (Gibco) for 30 min at 37°C with agitation. The resultant mononuclear cells were then stained with the following antibodies: CD31-PECy7 (BD Biosciences 561410, San Jose, California), CD45-PECy7 (BD Biosciences 552848), Sca1-FITC (BD Biosciences 562058), Integrin α7 (ITGA7)-Alexa Fluor 647 (AbLab, Vancouver, Canada) and VCAM-PE (Invitrogen RMCD10604). FACS was performed using a FACSAria II Cell Sorter (BD Biosciences) and SCs were collected according to the following sorting criteria: CD31-/CD45-/Sca1-/ITGA7+/VCAM+. FACs-purified SCs were plated at 3000 cells per well in eight-well Permanox chamber slides (Nunc Lab-Tek, Carlsbad, California) and cultured for five days in DMEM with 10% Horse Serum (Thermo Fisher Scientific, Carlsbad, California) and 5 ng/mL FGF2 (Cell Signaling Technologies, Danvers, Massachusetts).
6
Isolation of Aortic Endothelial Cells
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Four aortic arteries from four adult healthy male Beagle dogs were donated from Elanco Animal Health, Monheim, Germany. Arteries were kept at 4 °C in sterile 0.9% HBSS-HEPES buffer (pH 7.4; Gibco) supplemented with 1% penicillin (500 U/mL; Sigma-Aldrich) and streptomycin (500 µg/mL; Sigma-Aldrich, Darmstadt, Germany). For isolation of aortic endothelial cells, 0.025% collagenase type II (Worthington Biochemical Corporation, Lakewood, NJ, USA) was infused into the vessel lumen, the aorta was ligated with clamps and incubated for 20 min at 37 °C in 5% CO2 atmosphere. After gently massaging aortas, infused collagenase II-cell suspension was collected and immediately supplemented with 1 mL sterile fetal calf serum (FCS; Gibco, Langenselbold, Germany) to inactivate collagenase II. After two washing steps (400× g, 10 min, 4 °C), the cells were suspended in endothelium cell growth medium (ECGM; PromoCell, Heidelberg, Germany), plated in 25 cm2 plastic culture flasks (Nunc, Roskilde, Denmark) and kept at 37 °C in 5% CO2 atmosphere until reaching confluent cell layers. Culture medium was changed every 2–3 days.
7
Isolation of Satellite Cells and FAPs from Mice
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SCs were isolated from hindlimb muscles of C57BL/6 mice (n=4–6) using negative selection MACS Satellite Cell Isolation Kit (Miltenyi, 130–104-268) according to manufacture’s protocols. Muscles were minced and incubated with Muscle Dissociation Buffer (Ham’s F-10 (Sigma, N6908), 5% horse serum (Gibco, 16050–130), 1% penicillin/streptomycin (P/S) (Gibco, 15140–122), collagenase II (Gibco, 17101–105)) at 37°C for 60 min with agitation (60–70 RPM). Suspensions were re-incubated with collagenase II and dispase (Gibco, 17105–041) in Ham’s F-10 supplemented with 5% horse serum and 1% P/S at 37°C with agitation for 30 min. Suspensions were filtered and MACS LS column sorted (Miltenyi, 130–042-401). Cells were re-suspended in DMEM F-12 (Gibco, 31331–028) supplemented with 20% FBS (Gibco, 10270–106), 2% Ultroser G (Pall Gelman Sciences, 15950–017) and 1% P/S. FAPs were isolated from gastrocnemius muscles of n=4–6 juvenile C57BL/6 (4 d post-CTX), juvenile (7-week-old) D2-mdx, or adult (22-week-old) D2-mdx by pre-plating the cell suspension for 4 h after the digestion procedure described above. Cells were washed with PBS and left to amplify in non-coated flasks for 4–5 days in growth medium (DMEM F-12, 10% FBS, 1% P/S).
8
Immune Cell Isolation from Skeletal Muscle
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Isolation of immunocytes from skeletal muscle was performed as previously described (11) . Briefly, hindlimb muscles were dissected, minced and incubated for 30 min at 37°C in digestion buffer (Dulbecco's Modified Eagle Medium [DMEM], 2% fetal calf serum [FCS], collagenase II [2 mg/mL; Gibco] and DNase I [100 μg/mL; Sigma-Aldrich]). Immunocytes were enriched by density gradient centrifugation. The interphase between 40% and 80% Percoll (GE Healthcare) layers was recovered, washed, and stained for cytofluorometric analysis. For Foxp3-DTR experiments, a harsher isolation protocol was implemented (51) to increase recovery of myeloid cells, as done previously (52) . Briefly, hindlimb muscles were minced and incubated in digestion buffer (Ham's F10, 10% horse serum, collagenase II [800 U/mL; Gibco]) for 90 min at 37°C. Partially digested tissue and cells were sedimented by centrifugation, followed by further digestion using collagenase II (80 U/mL) and dispase (1 U/mL) for 30 min at 37°C. Fully digested tissue was mechanically disrupted by passage through a syringe and then filtered through a cell strainer. Isolated cells were washed and stained for cytofluorometric analysis.
9
Retrograde Labeling of Colon Neurons
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Healthy and CVH mice of 16 weeks of age were anesthetized with halothane and following midline laparotomy, three 10 μL injections of the fluorescent retrograde neuronal tracer cholera toxin subunit B conjugated to AlexaFluor-488 were made sub-serosally within the wall of the descending colon. Four days after injection mice were sacrificed by CO2 inhalation and DRGs from T10–L1 were surgically removed. DRGs were digested with 4 mg/mL collagenase II (GIBCO, Invitrogen) and 4 mg/mL dispase (GIBCO) for 30 min at 37°C, followed by 4 mg/mL collagenase II for 10 min at 37°C. Neurons were mechanically dissociated into a single-cell suspension via trituration through fire-polished Pasteur pipettes. Neurons were resuspended in DMEM (GIBCO) containing 10% FCS (Invitrogen), 2 mM L-glutamine (GIBCO), 100 μM MEM non-essential amino acids (GIBCO) and 100 mg/ml penicillin/streptomycin (Invitrogen). Neurons were spot-plated on 8 mm HCl treated coverslips coated with poly-D-lysine (800 μg/ml) and laminin (20 μg/ml) and maintained in an incubator at 37°C in 5% CO2.
10
Single-Cell RNA-Seq of Rat Skeletal Muscle
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Cell isolations were performed as previously described in mice [37 (link)] and modified slightly for rats [28 (link)]. Briefly, the gastrocnemius muscles from WB and HS male rats were excised and placed in muscle dissociation media (MDM) (Hams F-10 (Gibco, USA), 10% Horse Serum (Thermo Fisher), 1% penicillin/streptomycin (Gibco), 800 U/ml Collagenase II (Gibco)), and minced using sterilized surgical equipment. The muscle homogenate was then incubated in MDM for 1 h at 37 °C with gentle agitation. Following incubation, samples underwent further incubation in 1000 U/ml Collagenase II (Gibco) and 11 U/ml dispase (Gibco) for 30 min at 37 °C. The single-cell suspension was passed through an 18-gauge needle approximately 10 times prior to 0.2-μm filtration. Single cells were incubated in propidium iodide to identify dying/dead cells for removal via fluorescence-activated cell sorting (Sony Biotechnology, USA). Single-cell suspensions from each group were added to a Chromium Controller (10X Genomics, USA) using the Single Cell 3’ Reagent Kit per manufacturer’s instructions and sequenced on an Illumina HiSeq platform (Novogene, USA), yielding 200 million reads/sample.
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