Satellite cells were sorted based on established methods81 (link),82 (link). Briefly, entire hindlimb muscles from mice were digested with collagenase II (LS004177, Worthington, 1000 units per 1 ml) for 90 min at 37 °C, the digested muscles were then washed in washing medium (Ham’s F-10 medium (N6635, Sigma) containing 10% horse serum, heat-inactivated (HIHS, 26050088, Gibco, 1% P/S) before SCs were liberated by treating with Collagenase II (100 units per 1 ml) and Dispase (17105-041, Gibco, 1.1 unit per 1 ml) for 30 min. The suspensions were passed through a 20G needle to release myofiber-associated SCs. Mononuclear cells were filtered with a 40-µm cell strainer and sorted by BD FACSAria IV (fluorescence-activated cell sorting) with the selection of the positive GFP fluorescence signal. BD FACSVerse flow cytometer, BD FACSAria Fusion Cell Sorter and BD FACSDiva (Version 8.0.1, BD Biosciences) were used for the acquisition of flow cytometry data. Coverslips and cultural wells were coated with poly-D-lysine solution (p0899, Sigma) at 37 °C for overnight and then coated with extracellular matrix (ECM) (E-1270, Sigma) at 4 °C for at least 6 h. FACS-isolated SCs were seeded in coated wells and cultured in Ham’s F10 medium with 10% HIHS, 5 ng ml−1 β-FGF (PHG0026, Thermo Fisher Scientific) and 1% P/S.
Ls004177
Manufactured by Worthington
Sourced in United States
LS004177 is a lab equipment product. It is a precision instrument designed for laboratory applications. The core function of this product is to perform specific tasks required in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Facsaria fusion cell sorter, by BD (3 mentions)
Ham s f 10 medium, by Merck Group (3 mentions)
Facsaria 4, by BD (3 mentions)
E1270, by Merck Group (3 mentions)
Poly d lysine solution, by Merck Group (3 mentions)
Facsdiva, by BD (3 mentions)
Facsverse flow cytometer, by BD (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Cell strainer, by BD (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Sh800 sorter, by Sony (1 mentions)
Hemavet 950fs, by Drew Scientific (1 mentions)
Elastase, by Worthington (1 mentions)
E 0258, by Merck Group (1 mentions)
15 protocols using ls004177
1
Isolation and Culture of Satellite Cells
2
Isolation and Characterization of Endothelial Cells
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HUVECs were detached from cell culture plates using trypsin-EDTA (25300054, Thermo Fisher Scientific, Waltham, MA). Mouse embryos were minced and incubated with 0.5 mg/ml collagenase type II (LS004177, Worthington, Lakewood, CA) for 45 min at 37°C. Tissue suspensions were mashed twice through cell strainers (BD Biosciences; 100 and 40 μm). Endothelial cells were enriched by CD31 magnetic beads. Cells were suspended (106 cells/ml) and incubated with different fluorophores coupled to primary antibodies against DLL1 (FAB1818A, R & D Systems), DLL4 (FAB1506A, R & D Systems), Dll4 (563802, BD Biosciences), CD34 (553733, BD Biosciences) for 20 min on ice. Concentration of the different antibodies was determined by titration, in order to get optimal compensation during acquisition.
3
Skin wound digestion protocol
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The dorsal skin of 8 weeks old FVB/n mice was shaved and cleaned with 70% ethanol under anaesthesia, and the skin was incised up to the level of the subcutaneous adipose tissue with a 5 mm Acu-Punch biopsy tool. Five days later, the wounded skin and control skin from the same animal were excised and digested into single cell suspension5 (link). Briefly, wounded and control skin were washed in PBS, minced thoroughly with scissors, and incubated for 15 min with DMEM supplemented with 0.1% collagenase II (Worthington, LS4176) and 0.1% collagenase IV (Worthington, LS004177) on stir plate in 37 °C water bath.
4
Cardiac Fibroblast Isolation from Mouse Hearts
5
Cardiac Fibroblast Isolation from Mouse Hearts
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Hearts were excised from ten 8-week-old male and female C57Bl/6J mice and the ventricles and septum were isolated, rinsed in ice-cold PBS and minced into small pieces using sterile micro-scissors. Tissue fragments were digested in DMEM + 2% BGS and 1% penicillin-streptomycin containing type 2 collagenase (100 units/mL; LS004177, Worthington, USA) for 20 minutes at 37 °C under gentle agitation. The digested tissue was triturated repeatedly to promote tissue dissociation. Dense fragments settled for 2 minutes and the supernatant, containing the cardiac fibroblasts, was collected and spun at 1,000 g for 5 minutes. The cell pellet was resuspended in 10 mL of DMEM + 10% BGS and 1% penicillin-streptomycin and kept on ice. This process was repeated 3 times until all the tissue was adequately digested. To remove cardiomyocytes and cell debris, cell suspensions were spun at 300 g, followed by centrifugation of the supernatant at 1,000 g. The final cell pellet containing the cardiac fibroblasts was resuspended in DMEM + 10% BGS and 1% penicillin-streptomycin and pre-incubated on 0.1% gelatin-coated plates for 2 hours to allow fibroblast adherence before replenishment of the cell culture medium.
6
Isolation and Culture of Valve Interstitial Cells
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Human VICs were isolated as previously described47 . Briefly, the excised aortic valve leaflets were treated with 1 mg/ml collagenase II (LS004177, Worthington Biochemical Corporation) for 10 minutes and endothelial cells were scraped off from both sides of the leaflets. Then the leaflets were subjected to digestion with 1 mg/ml collagenase II overnight at 37 °C. The cell suspension was homogenized by pipetting and centrifuged at 300 × g for five minutes. VICs were cultured in standard growth medium (DMEM (41966-052, Gibco) supplemented with 10% FBS (HyClone, SH30070.03, GE Healthcare) and 50 µg/mL of gentamicin (15750-037, Gibco) at 37 °C in 5% CO2 until confluence of 70–80% before passaging. Cells from passages 2 to 6 were used for all experiments.
7
Isolation of Ventricular Myocytes from Mice
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To isolate ventricular myocytes, 2-weeks-old WT and cKO littermates were used. Ventricular myocytes were isolated by perfusion with a Ca2+-free normal Tyrode solution containing collagenase (Worthington, type 2, LS004177) on a Langendorff column at 37 °C as previously described52 (link) with minor modifications. Isolated ventricular myocytes were kept in high K+, low Cl− solution at 4 °C until use. Normal Tyrode solution contained (in millimoles per liter) 140 NaCl, 5 KCl, 1 MgCl2, 1.8 CaCl2, 10 Hepes, and 10 glucose, adjusted to pH 7.4 with NaOH. The Ca2+-free solution contained (in millimoles per liter) 140 NaCl, 5 KCl, 1 MgCl2, 10 Hepes, and 10 glucose, adjusted to pH 7.4 with NaOH. The high K+, low Cl− solution contained (in millimoles per liter) only Ca2+-tolerant, rod-shaped myocytes with cross-striations and without spontaneous contractions or significant granulation were selected for electrophysiological experiments.
8
Isolation and Culture of Satellite Cells
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Satellite cells were sorted based on established methods81 (link),82 (link). Briefly, entire hindlimb muscles from mice were digested with collagenase II (LS004177, Worthington, 1000 units per 1 ml) for 90 min at 37 °C, the digested muscles were then washed in washing medium (Ham’s F-10 medium (N6635, Sigma) containing 10% horse serum, heat-inactivated (HIHS, 26050088, Gibco, 1% P/S) before SCs were liberated by treating with Collagenase II (100 units per 1 ml) and Dispase (17105-041, Gibco, 1.1 unit per 1 ml) for 30 min. The suspensions were passed through a 20G needle to release myofiber-associated SCs. Mononuclear cells were filtered with a 40-µm cell strainer and sorted by BD FACSAria IV (fluorescence-activated cell sorting) with the selection of the positive GFP fluorescence signal. BD FACSVerse flow cytometer, BD FACSAria Fusion Cell Sorter and BD FACSDiva (Version 8.0.1, BD Biosciences) were used for the acquisition of flow cytometry data. Coverslips and cultural wells were coated with poly-D-lysine solution (p0899, Sigma) at 37 °C for overnight and then coated with extracellular matrix (ECM) (E-1270, Sigma) at 4 °C for at least 6 h. FACS-isolated SCs were seeded in coated wells and cultured in Ham’s F10 medium with 10% HIHS, 5 ng ml−1 β-FGF (PHG0026, Thermo Fisher Scientific) and 1% P/S.
9
Isolation and Purification of Muscle Stem Cells
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Isolation of single cell and MuSC by physical dissociation and enzymatic digestion was followed as previously described (Liu et al., 2015 (link)). In brief, we dissected the hind limb muscles from the mouse and digested the minced muscles with collagenase II (800 U/ml [LS004177; Worthington,]) in wash medium (F10, 10% horse serum, 1% P/S) for 90 min. The tissue was further digested by collagenase II (100 U/ml) and dispase (1.1 U/ml) in wash medium for 30 min and passed 10 times through a 20-gauge needle, resuspended with wash medium, and went through a 40 μm filter to get single-cell suspension for cell sorting. Single-cell suspensions from WT mice were stained with Propidium Iodide (PI, 1:1,000), and then was sorted on a Sony SH800 sorter equipped with 405-, 488-, 561-, and 633-nm lasers. PI− single cell from WT mice was sorted to single-cell RNAseq. GPF+; PI− or YFP+; PI− single cell from reporter mice was used to isolate MuSC. For MuSCs transplantation, single-cell suspension was stained with BETAGLYCAN antibody (MA5-17187; 1:200; Invitrogen) and stained with APC/Cy7 Goat anti-mouse IgG (405316; 1:500; BioLegend). APC/Cy7 Goat anti-mouse IgG Purified Mouse IgG2b, κ Isotype Ctrl Antibody (400302; 1:500; BioLegend) or secondary single-stained was used as control, the BETAGLYCAN+; GPF+; PI− and BETAGLYCAN−; GPF+; PI− were sorted to transplant, respectively.
10
Isolation and Culture of Satellite Cells
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Satellite cells were sorted based on established methods81 (link),82 (link). Briefly, entire hindlimb muscles from mice were digested with collagenase II (LS004177, Worthington, 1000 units per 1 ml) for 90 min at 37 °C, the digested muscles were then washed in washing medium (Ham’s F-10 medium (N6635, Sigma) containing 10% horse serum, heat-inactivated (HIHS, 26050088, Gibco, 1% P/S) before SCs were liberated by treating with Collagenase II (100 units per 1 ml) and Dispase (17105-041, Gibco, 1.1 unit per 1 ml) for 30 min. The suspensions were passed through a 20G needle to release myofiber-associated SCs. Mononuclear cells were filtered with a 40-µm cell strainer and sorted by BD FACSAria IV (fluorescence-activated cell sorting) with the selection of the positive GFP fluorescence signal. BD FACSVerse flow cytometer, BD FACSAria Fusion Cell Sorter and BD FACSDiva (Version 8.0.1, BD Biosciences) were used for the acquisition of flow cytometry data. Coverslips and cultural wells were coated with poly-D-lysine solution (p0899, Sigma) at 37 °C for overnight and then coated with extracellular matrix (ECM) (E-1270, Sigma) at 4 °C for at least 6 h. FACS-isolated SCs were seeded in coated wells and cultured in Ham’s F10 medium with 10% HIHS, 5 ng ml−1 β-FGF (PHG0026, Thermo Fisher Scientific) and 1% P/S.
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