Muscles were dissected from mice and dissociated mechanically. All hindlimb muscles were used except in experiments where FAPs were isolated from VMOs injected into TA muscles. In this case, only the TA was dissected. The muscle suspension was digested using Collagenase II (760 U/ml; Worthington Biochemical Corporation) in Ham’s F10 (Invitrogen) with 10% horse serum (Invitrogen) for 90 minutes at 37°C with agitation. The suspension was then washed and digested in Collagenase II (152 U/ml; Worthington Biochemical Corporation) and dispase (2 U/ml; Invitrogen) for 30 minutes at 37°C with agitation. The resultant mononuclear cells were then stained with the following antibodies: VCAM-1-biotin (clone 429; BioLegend, 105704), CD31-APC (clone MEC 13.3; BioLegend, 102510), CD45-APC (clone 30-F11; BioLegend, 103112) and Sca-1-Pacific Blue (clone D7; BioLegend, 108120) at 1:75. Streptavidin-PE-Cy7 (BioLegend, 405206) at 1:75 was used to amplify the VCAM-1 signal. Fluorescence-activated cell sorting (FACS) was performed using BD-FACS Aria II and BD-FACS Aria III cell sorters equipped with 488 nm, 633 nm and 405 nm lasers. The cell sorters were carefully optimized for purity and viability and sorted cells were subjected to FACS analysis immediately post-sorting to confirm FAP purity.
Cd31 apc clone mec 13.3
Manufactured by BioLegend
CD31-APC (clone MEC 13.3) is a fluorochrome-conjugated monoclonal antibody that recognizes the CD31 (PECAM-1) antigen. CD31 is a cell adhesion molecule expressed on the surface of various cell types, including endothelial cells, platelets, and some leukocyte subsets.
Automatically generated - may contain errors
Lab products found in correlation
Cd45 apc clone 30 f11, by BioLegend (3 mentions)
Facsaria 2, by BD (2 mentions)
Facsaria 3, by BD (2 mentions)
Ham s f10, by Thermo Fisher Scientific (2 mentions)
Streptavidin pe cy7, by BioLegend (2 mentions)
Horse serum, by Thermo Fisher Scientific (2 mentions)
Dispase, by Thermo Fisher Scientific (2 mentions)
Collagenase 2, by Worthington (2 mentions)
Vcam 1 biotin clone 429, by BioLegend (2 mentions)
Sca 1 pacific blue clone d7, by BioLegend (2 mentions)
Facscanto 2, by BD (1 mentions)
Triton x 100, by Thermo Fisher Scientific (1 mentions)
Collagenase type 4, by Worthington (1 mentions)
Daf 2 da, by Abcam (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Moflo xdp flow cytometer, by Beckman Coulter (1 mentions)
Hyaluronidase, by Merck Group (1 mentions)
Co2 independent medium, by Thermo Fisher Scientific (1 mentions)
Collagenase a, by Roche (1 mentions)
Dispase, by Roche (1 mentions)
4 protocols using cd31 apc clone mec 13.3
1
Isolation and Purification of Muscle-Derived Fibro-Adipogenic Progenitors
2
Isolation and Purification of Muscle-Derived Fibro-Adipogenic Progenitors
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Muscles were dissected from mice and dissociated mechanically. All hindlimb muscles were used except in experiments where FAPs were isolated from VMOs injected into TA muscles. In this case, only the TA was dissected. The muscle suspension was digested using Collagenase II (760 U/ml; Worthington Biochemical Corporation) in Ham’s F10 (Invitrogen) with 10% horse serum (Invitrogen) for 90 minutes at 37°C with agitation. The suspension was then washed and digested in Collagenase II (152 U/ml; Worthington Biochemical Corporation) and dispase (2 U/ml; Invitrogen) for 30 minutes at 37°C with agitation. The resultant mononuclear cells were then stained with the following antibodies: VCAM-1-biotin (clone 429; BioLegend, 105704), CD31-APC (clone MEC 13.3; BioLegend, 102510), CD45-APC (clone 30-F11; BioLegend, 103112) and Sca-1-Pacific Blue (clone D7; BioLegend, 108120) at 1:75. Streptavidin-PE-Cy7 (BioLegend, 405206) at 1:75 was used to amplify the VCAM-1 signal. Fluorescence-activated cell sorting (FACS) was performed using BD-FACS Aria II and BD-FACS Aria III cell sorters equipped with 488 nm, 633 nm and 405 nm lasers. The cell sorters were carefully optimized for purity and viability and sorted cells were subjected to FACS analysis immediately post-sorting to confirm FAP purity.
3
Quantification of Endothelial Nitric Oxide
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Isolated lower limb muscle was minced and digested with collagenase type IV (Worthington; final concentration, 2 mg/mL) and 2 mM CaCl2 in PBS at 37°C for 45 minutes. Cells were fixed using 0.1% PFA for 10 minutes at room temperature (RT), followed by permeabilization using 0.1% Triton X-100 (Thermo Fisher Scientific) for 5 minutes at RT. Unspecific binding was blocked using FcR receptor blocking reagent (Miltenyi Biotec; 130-092-575), followed by incubation with allophycocyanin-labeled primary antibodies against CD31 (CD31-APC; clone MEC13.3; BioLegend; 102510; 1:100). To visualize NO, 1 × 106 unfixed cells were incubated with fluorescent NO probe 1.5 μM DAF-2 DA (Abcam; ab146631) at 37°C for 35 minutes, based on preliminary dose-finding studies and as published previously (69 (link)). Cells were washed and immediately analyzed using flow cytometry (BD Biosciences FACSCanto II). Forward and side scatter gates were set to exclude debris and cellular aggregates. Unstained cells were used as a negative controls.
4
Isolation of Mammary Epithelial Cell Subsets
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Mammary epithelial cells (MECs) and the separation of basal and luminal cells were performed as described elsewhere (Stingl et al., 2006 (link); Taddei et al., 2008 (link)). Once mechanically dissociated, mammary fat pads were digested (90 min, 37°C) in CO2-independent medium (Invitrogen) containing 5% FBS, 3 mg/ml collagenase A (Roche Diagnostics) and 100 U/ml hyaluronidase (Sigma). Cells were resuspended in 0.25% trypsin-EDTA (1 min), and then in 5 mg/ml dispase (Roche Diagnostics) with 0.1 mg/ml DNase I (Sigma) (5 min). Red blood cells were lysed with 0.17 mM NH4Cl. Basal and luminal cells were isolated from mammary epithelial cells obtained from the inguinal glands. Cells were stained with the following antibodies: CD24-PerCP-Cy5.5 (clone M1/69; BD Pharmingen), CD49f-PE (clone GoH3; BD Pharmingen), CD45-APC (clone 30-F11; Biolegend) and CD31-APC (clone MEC13.3; Biolegend). Basal (CD24-low/CD49f-high) and luminal (CD24-high/CD49f-low) cells were purified using MoFlo XDP Flow Cytometer (Beckman Coulter).
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