ECs were isolated as previously described (Haber et al., 2017 (link)), with modifications. Briefly, the ileal, cecal, and colonic tissues of VB12- and PBS-gavaged mice were dissected and washed with cold PBS. The tissues were opened longitudinally and cut into small fragments (2–3 cm in length), followed by incubation with 30 mM EDTA-PBS on ice for 30 min, during which the tissues were shaken vigorously every 8–10 min. Isolated crypts were washed once with cold PBS and dissociated with TrypLE Express (Invitrogen) for 10 min at room temperature. The single-cell suspension was passed through a 70-μm cell strainer and used for FACS. To FACS-sort the cells, cell suspensions were labeled with a cocktail of fluorescent antibodies specific for APC-CD45 (catalog 17-0451-83; Invitrogen), FITC-CD31 (catalog 102406; BioLegend), PE/Cy7-TER-119 (catalog 116222; BioLegend), and PE-EpCAM (catalog 12-5791-83; Invitrogen). Dead cells were excluded using LIVE/DEAD Fixable Violet Dead Cell Stain (Invitrogen). CD45− CD31− TER-119− EpCAM+ ECs were sorted using a SONY SH800S Cell Sorter or a CytoFLEX SRT Cell Sorter.
Cd31 fitc
Manufactured by BioLegend
Sourced in United States
CD31-FITC is a fluorochrome-conjugated antibody that binds to the CD31 antigen, also known as PECAM-1 (Platelet Endothelial Cell Adhesion Molecule 1). CD31 is a cell surface glycoprotein expressed on endothelial cells, platelets, and certain leukocyte subsets. This product can be used for the identification and enumeration of CD31-positive cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd45 fitc, by BioLegend (9 mentions)
Cd90 fitc, by BioLegend (5 mentions)
Cd73 apc, by BioLegend (4 mentions)
Cd146 pe, by BioLegend (4 mentions)
Cd73 pe, by BioLegend (3 mentions)
Facsaria, by BD (3 mentions)
Cd105 pe, by Thermo Fisher Scientific (3 mentions)
Cd90 pe, by BioLegend (3 mentions)
Cd45 percp cy5, by BioLegend (3 mentions)
Sca1 apc, by BioLegend (3 mentions)
Cd11b apc, by BioLegend (3 mentions)
Facsaria 2, by BD (3 mentions)
Cd45 apc, by Thermo Fisher Scientific (2 mentions)
Pe cy7 ter 119, by BioLegend (2 mentions)
Epcam pe, by Thermo Fisher Scientific (2 mentions)
Live dead fixable violet dead cell stain, by Thermo Fisher Scientific (2 mentions)
Tryple express, by Thermo Fisher Scientific (2 mentions)
Cd45 pe cy7, by Thermo Fisher Scientific (2 mentions)
V450 sca1, by BD (2 mentions)
Efluor660 vcam1, by Thermo Fisher Scientific (2 mentions)
33 protocols using cd31 fitc
1
Isolation and FACS-Sorting of Intestinal Epithelial Cells
2
Isolation and Culture of viECs
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On day 7 of the viEC differentiation, cells were dissociated with Accutase and neutralized with cold Stempro-34 SFM medium. Cells were spun at 1,000 rpm for 5 min and then washed with MACS buffer containing DPBS with 0.5% BSA and 2 mM EDTA. Cells were resuspended in 200 μL MACS buffer and co-stained for 30 min on ice with 10 μL pre-conjugated FITC CD31 (BioLegend) antibody and PE CD144 (BioLegend) antibody. Cells were rinsed once with MACS buffer and sorted on a Beckman Coulter Astrios Sorter at the Duke Flow Cytometry Shared Resource Facility. Cells were then replated on collagen-coated plates and cultured in viEC-conditioned medium during passage 0 directly after sorting. Media were changed every other day. viECs were routinely passaged at 80%–90% confluency using Accutase onto collagen-coated plates and continuously cultured in viEC medium consisting of Stempro-34 SFM medium with 10% HI-FBS, 50 ng/mL VEGF165, and 2 μg/mL heparin after passage 1. HGPS and normal viECs were used between passages 1 and 4.
3
Quantifying Nck1 Impact on BM-MSCs
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To assess the BM-MSCs count, cells were isolated from Nck1+/+ and Nck1−/− mice as described above and used for flow cytometry analysis at passage 3. BM-MSCs were dissociated using a non-enzymatic dissociation buffer and resuspended in PBS/0.1%BSA. BM-MSCs were stained with the following anti-mouse antibodies: FITC CD31 ( Clone: 390; BioLegend 102405), PerCP/Cy5.5 CD45 ( Clone: 30-F11; BioLegend 103131), Pacific Blue Sca-1 (clone D7; BioLegend 108119), and PE CD140a (PDGFRα) ( Clone: APA5; BioLegend 135905) for 1 h at 4 °C. The stained BM-MSCs were then sorted using a BD FACSCanto II flow cytometer. Data was quantified and analyzed using FACSDiva.
4
Stromal Vascular Cell Characterization
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Freshly isolated stromal vascular cells prepared from paired samples of Om and Abdsc adipose tissues were used for flow cytometry (27 (link)). Antibodies were purchased from eBioscience (FITC CD45, FITC CD14, FITC CD31, and Cy5 CD34) and BioLegend (phycoerythrin CD29, and brilliant blue CD90). Total stromal and sorted cells were used for RNA extraction and quantitative PCR measurement of cell markers or plated for testing adipogenesis.
5
Isolation of Muscle Stem Cells
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MuSCs were FACS-sorted from hind limb muscles in postnatal 1-week mice according to the reported protocol [45 (link)]. Briefly, mechanical and enzymatic dissociation of cells, released resident mononucleated cells, then followed by antibody staining and FACS isolation.
Mononuclear cells were incubated with antibodies against CD45 (PE/Cy7-CD45, eBioscience, 25-0451-82), CD31 (FITC-CD31, BioLegend, 102506), Sca1 (V450-Sca1, BD, 560653) and VCAM1 (efluor660-VCAM1, eBioscience, 50-1061-80). Immunostained cells were briefly washed, passed through a 70-μM nylon mesh (Falcon) and suspended at a concentration of 106–107 cells per mL. Cells were further separated with the Beckman Coulter MoFlo XDP system. Sorting gates were strictly defined on the basis of control cells stained with single antibodies as well as the patterns of forward scatter and side scatter of MuSCs in preliminary tests. Collection of the cells that were positive for VCAM1 expression and negative for CD31, CD45, and Sca1.
Mononuclear cells were incubated with antibodies against CD45 (PE/Cy7-CD45, eBioscience, 25-0451-82), CD31 (FITC-CD31, BioLegend, 102506), Sca1 (V450-Sca1, BD, 560653) and VCAM1 (efluor660-VCAM1, eBioscience, 50-1061-80). Immunostained cells were briefly washed, passed through a 70-μM nylon mesh (Falcon) and suspended at a concentration of 106–107 cells per mL. Cells were further separated with the Beckman Coulter MoFlo XDP system. Sorting gates were strictly defined on the basis of control cells stained with single antibodies as well as the patterns of forward scatter and side scatter of MuSCs in preliminary tests. Collection of the cells that were positive for VCAM1 expression and negative for CD31, CD45, and Sca1.
6
Stem Cell Surface Marker Analysis
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Flow cytometric analysis was carried out to detect stem cell surface markers and PDLSCs at passage 3 were used.19 (link) After being digested with 0.25% trypsin containing 0.02% EDTA (Gibco, USA), the cells were washed with PBS once and adjusted to single-cell suspensions with a density of 1 × 106 cells per mL. Subsequently, 100 μL of the suspension was respectively added into 5 microtubes, and 1 μL of the following antibodies were added to the relevant tubes: PE-CD73 (Biolegend, San Diego, USA); PE-CD105 (Biolegend, San Diego, USA); FITC-CD31 (Biolegend, San Diego, USA); and FITC-CD45 (Biolegend, San Diego, USA). Cell suspensions without added antibodies served as controls. All of the microtubes were kept away from light and incubated for 15 min at room temperature. Finally, 300 μL of PBS supplemented with 2% FBS was added to the tubes, and the cells were then resuspended gently and adequately. The flow cytometry we used was Beckman CytoFLEX FCM (Beckman Coulter, USA) and the obtained data was analysed by Flowjo v9.3.2.
7
Flow Cytometry Analysis of Lung Cell Phenotypes
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The digested cells from lung tissues or cultured cells were respectively labeled with a flow cytometric antibody against FITC-CD31(1:100, BioLegend), FITC-CD90 (1:100, BioLegend), FITC-Vimentin (1:100, BioLegend), and APC-CY7-CD45 (1:100, BioLegend) and incubated at 37 °C for 10 min in the dark. Subsequently, the sections were washed with PBS containing 10% FBS. The cells were then analyzed via high-resolution flow cytometry (BD FACSAria, BD bioscience, USA).
For FACS data analysis, cells were sorted using 488 nm and 633 nm lasers, and the fluorescence signals were captured on FITC, CY-7, FSC (forward scatter), and SSC (sider scatter) channels. In order to ensure the viability of cells, 2000 events were recorded for each sample at a rate of 0.5 μl/s. Cytometry data were processed to generate the percentage values of positive events using FlowJo (version 10.4) software (Becton, Dickinson & Company, USA).
For FACS data analysis, cells were sorted using 488 nm and 633 nm lasers, and the fluorescence signals were captured on FITC, CY-7, FSC (forward scatter), and SSC (sider scatter) channels. In order to ensure the viability of cells, 2000 events were recorded for each sample at a rate of 0.5 μl/s. Cytometry data were processed to generate the percentage values of positive events using FlowJo (version 10.4) software (Becton, Dickinson & Company, USA).
8
Isolation of Intestinal Epithelial Cells
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The ileum (similar length as colon moving proximal from the ileocecal junction), cecum and colon were dissected from mice after MCAO or sham surgery. After washing with cold PBS, the tissues were opened longitudinally and cut into small fragments (2–3 cm in length), followed by incubation with 30 mM EDTA-PBS on ice for 30 min, during which the tissues were shaken vigorously every 8–10 min. Isolated crypts were washed once with cold PBS and dissociated using TrypLE Express (Invitrogen) for 10 min at room temperature. Cell suspensions were passed through a 70-μm cell strainer and then labeled with a cocktail of fluorescent antibodies specific for APC-CD45 (Invitrogen, catalog 17-0451-83), FITC-CD31 (BioLegend, catalog 102406), PE/Cy7-TER-119 (BioLegend, catalog 116222), and PE-EpCAM (Invitrogen, catalog 12-5791-83). Dead cells were excluded using LIVE/DEAD Fixable Violet Dead Cell Stain (Invitrogen). CD45− CD31− TER-119- EpCAM+ ECs were sorted using a SONY SH800S Cell Sorter.
9
Isolation of Skeletal Muscle Stem Cells
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MuSCs were FACS-sorted from hind limb muscles of postnatal 1-week mice as previously described (Xie et al., 2018 (link)). Briefly, mechanical and enzymatic dissociation of cells, released resident mononucleated cells, then followed by antibody CD45 (PE/Cy7-CD45, eBioscience, 25-0451-82), CD31 (FITC-CD31, BioLegend, 102506), Sca1 (V450-Sca1, BD, 560653) and VCAM1 (efluor660-VCAM1, eBioscience, 50-1061-80) staining and FACS isolation. Collection of the cells those were positive for VCAM1 expression and negative for CD31, CD45, and Sca1.
10
Isolation of Adipocytes and Stromal Vascular Cells
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