Epididymal and inguinal fat pads were cut in small pieces and incubated with 1 mg/ml collagenase I for 40 min44 (link),45 (link). The cell suspension was filtered through a 150 μm nylon mesh, and the stromalvascular fraction (SVF) was isolated by low-speed centrifugation. For FACS analysis, erythrocyte-free SVF cells were incubated with a mix of antibodies against different surface markers as described previously46 (link) (anti-Ter119 FITC, eBioscience, 11-5921, 0.5 mg.ml−1; anti-Cd45, FITC, eBioscience, 11-0451, 0.5 mg.ml−1; anti-Cd31 FITC, eBioscience, 11-0311, 0.5 mg.ml−1; anti-Sca1 PE/Cy7, BioLegend / Biozol, 122514, 1/500; and anti-Cd34 PE, abcam, ab23830, 1/250) and sorted using an Aria flow cytometer (BD Biosciences). Dead cells were removed using DAPI staining (1/10000). Cells negative for Ter119, Cd45, and Cd31 and positive for both Sca1 and Cd34 were considered as adipocyte precursor cells. Primary adipocytes were cultured in DMEM/F12 containing 10% foetal bovine serum (FBS).
Anti sca1 pe cy7
Manufactured by BioLegend
Sourced in United States
Anti-Sca1 PE/Cy7 is a fluorochrome-conjugated antibody that binds to the Sca1 (Stem Cell Antigen-1) marker. Sca1 is a glycosylphosphatidylinositol (GPI)-anchored cell surface protein commonly used as a marker for hematopoietic stem and progenitor cells.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Thermo Fisher Scientific (3 mentions)
Anti cd45 apc cy7, by BioLegend (2 mentions)
Anti pdgfra cd140a pe, by BioLegend (2 mentions)
Lsr 2, by BD (2 mentions)
Facsdiva, by BD (2 mentions)
Red blood cell lysis buffer, by BD (2 mentions)
Anti c kit apc, by BioLegend (2 mentions)
Anti cd31 fitc, by Thermo Fisher Scientific (1 mentions)
Anti cd45 fitc, by Thermo Fisher Scientific (1 mentions)
Anti cd34 pe, by Abcam (1 mentions)
Anti ter119 fitc, by Thermo Fisher Scientific (1 mentions)
Aria flow cytometer, by BD (1 mentions)
Anti cd34 fitc, by Thermo Fisher Scientific (1 mentions)
Fortessa flow cytometer, by BD (1 mentions)
Sytox blue, by Thermo Fisher Scientific (1 mentions)
Anti ter119 apc, by BioLegend (1 mentions)
Anti c kit apc cy7, by Thermo Fisher Scientific (1 mentions)
Anti cd150 apc, by BioLegend (1 mentions)
Facsaria, by BD (1 mentions)
Ter119, by BioLegend (1 mentions)
6 protocols using anti sca1 pe cy7
1
Isolation and Characterization of Adipocyte Precursor Cells
2
Fluorescence-Activated Cell Sorting of Adipose Stromal Cells
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For fluorescence‐activated cell sorting (FACS) analysis, SVFs were resuspended in red blood cell lysis buffer (BD Biosciences) for 15 min at room temperature and followed by cell surface marker staining using anti‐PDGFRa (CD140a)‐PE (rat, 1:200; BioLegend, San Diego, CA, USA), anti‐Sca1‐PE/Cy7 (rat, 1:300; BioLegend), and anti‐CD45‐APC/Cy7 (rat, 1:300; BioLegend). Cell sorting and analytic cytometry were performed using FASCAria (Becon Dickinson) and BD LSR II (BD Biosciences, San Jose, CA, USA) flow cytometers, respectively. All compensation was performed using single‐color controls in BD FACSDiVa (BD Biosciences) software at the time of acquisition. 100,000 stromal vascular cells were examined for each sample. Raw data were processed using FlowJo software (Tree Star, Ashland, OR, USA). Results of analytic cytometry are presented as the fraction of CD45−:Sca1+:PDGFRα+ per total cells analyzed.
3
Multicolor Flow Cytometry of Hematopoietic Cells
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Cells were isolated from BM by flushing marrow cavities. Cells were isolated from FL from timed pregnancies. Cells were analyzed on a Fortessa flow cytometer or sorted on a FACS Aria (BD). The following anti–mouse fluorescence-conjugated antibodies were used: anti-B220, CD3, Ter119, Mac-1, Gr-1 conjugated to Pacific blue as a lineage cocktail, anti–c-kit–APC-Cy7, anti-CD34–FITC, and anti-CD16/32–PerCp-Cy5.5 (eBioscience and Affymetrix); anti–Sca-1–PE-Cy7; anti-Ter119–APC and anti–Gr-1–PE; anti–Mac-1–PE-Cy5 and anti-CD150–APC (BioLegend); and anti–Mac-1–AF700, anti-CD48–PE, and anti-CD71–FITC (BD). Cells were counterstained for viability with Sytox blue or propidium iodide (Thermo Fisher Scientific). Viability for cell quantification was determined using a parallel tube stained with Sytox blue.
4
FACS Analysis of Stromal Vascular Fraction
5
Isolation and Characterization of Murine Hematopoietic Stem/Progenitor Cells
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Total bone marrow was isolated from bones (tibia, femur, pelvis and vertebral column). Cells were then stained with anti–c‐Kit‐APC (BioLegend; 105812) antibody at 4°C for 30 min. c‐Kit+ cells from total bone marrow (BM) were enriched through magnetic‐activated cell separation (MACS; Miltenyi Biotec) according to the manufacturer’s protocol (LS Column, 130‐042‐402; anti–APC‐microbeads, 130‐090‐855). Cells were incubated with a lineage cocktail containing biotinylated antibodies against CD4 (BioLegend; 100508), CD8 (BioLegend; 100704), TER‐119 (BioLegend; 116204), CD11b (BioLegend; 101204), Gr‐1 (BioLegend; 108404), and B220 (BioLegend; 103222) at 4°C for 30 min. After washing, cells were incubated with anti–Sca‐1‐PE‐Cy7 (BioLegend; 108114) and anti–c‐Kit‐APC (BioLegend; 105812) antibodies. Biotinylated antibodies were developed with streptavidin‐APC‐Cy7 (BioLegend; 405208). LSK cells, which contain hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs), were FACS sorted by selecting for Sca‐1+, c‐Kit+, lineage− cells.
6
Isolation and Characterization of Murine Hematopoietic Progenitor Cells
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Experimental procedures were approved by the Dier Experimenten Commissie of the Royal Netherlands Academy of Arts and Sciences and performed according to the guidelines.
Primary bone marrow cells were harvested from 3-months-old C57BL/6 mice. Femur and Tibia were extracted, the bones ends were cut away to access the bone marrow which was flushed out using a 22G syringe with HBSS (-Ca, -Mg, -phenol red, Gibco 14175053) supplemented with Pen-Strep and 1% FCS. The bone marrow was dissociated and debris was removed by passing it through a 70 µm cell strainer (Corning, 431751). Cells were washed with 25 ml supplemented HBSS before linage marker staining was performed following the instructions of the EasySep™ Mouse Hematopoietic Progenitor Cell Isolation Kit (Stemcell) at half of the recommended concentration of the biotinylated antibodies. This was followed by 30min incubation at 4 o C with a Streptavidin-PE (Biolegend, 1:5000), anti c-kit-APC (Biolegend, 1:800) and anti sca1-PeCy7 (Biolegend, 1:400). After 2 additional washes with HBBS (+PS, +FCS) cells were prepared following the sortChIC protocol for the 4 different histone modifications.
Primary bone marrow cells were harvested from 3-months-old C57BL/6 mice. Femur and Tibia were extracted, the bones ends were cut away to access the bone marrow which was flushed out using a 22G syringe with HBSS (-Ca, -Mg, -phenol red, Gibco 14175053) supplemented with Pen-Strep and 1% FCS. The bone marrow was dissociated and debris was removed by passing it through a 70 µm cell strainer (Corning, 431751). Cells were washed with 25 ml supplemented HBSS before linage marker staining was performed following the instructions of the EasySep™ Mouse Hematopoietic Progenitor Cell Isolation Kit (Stemcell) at half of the recommended concentration of the biotinylated antibodies. This was followed by 30min incubation at 4 o C with a Streptavidin-PE (Biolegend, 1:5000), anti c-kit-APC (Biolegend, 1:800) and anti sca1-PeCy7 (Biolegend, 1:400). After 2 additional washes with HBBS (+PS, +FCS) cells were prepared following the sortChIC protocol for the 4 different histone modifications.
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