DPSCs and aBMMSCs were characterized for mesenchymal (CD90, CD73, CD49f, CD146, STRO-1), endothelial (CD105), hematopoietic (CD45, CD34, CD117/c-kit) and embryonic (SSEA-1, SSEA-3, SSEA-4) stem cell (SC) markers at p.2–3, p.6–7 and p.10–11 by flow cytometry, as described previously [26 (link)], using the following fluorochrome-conjugated mouse anti-human antibodies: CD90-fluorescein isothiocyanate (FITC), CD73-phycoerythrin (PE), CD34-allophycocyanin (APC), STRO-1-FITC, CD146-PE, CD49f-APC, CD105-APC, SSEA-1-PE, SSEA-3-PE, SSEA-4-FITC, CD117-Peridinin-Chlorophyll-Protein-cyanin 5.5 (PerCP-Cy5.5) and CD45-PE (all BioLegend, Fell, Germany). Analysis was performed by means of a Guava® easyCyte 8HT Benchtop Flow Cytometer (Merck Millipore, Billerica, MA, USA). A total of 50,000 events were acquired for each sample. Data were analyzed using GuavaSoft 3.1.1 and Summit 5.1 software. In addition to determining the percentage of cells positive for each marker, the cell size and cell internal complexity (granularity) distribution profiles were analyzed by forward scatter (FSC) vs side scatter (SSC) fluorescence intensity plots, respectively.
Cd49f apc
Manufactured by BioLegend
Sourced in United States
CD49f-APC is a flow cytometry antibody produced by BioLegend. It binds to the CD49f (also known as alpha 6 integrin) cell surface antigen and is conjugated to the fluorescent dye APC (allophycocyanin).
Automatically generated - may contain errors
Lab products found in correlation
Epcam fitc, by BioLegend (2 mentions)
Ssea4 fitc, by BioLegend (1 mentions)
Cd146 pe, by BioLegend (1 mentions)
Cd45 pe, by BioLegend (1 mentions)
Stro 1 fitc, by BioLegend (1 mentions)
Cd105 apc, by BioLegend (1 mentions)
Guava easycyte 8ht benchtop flow cytometer, by Merck Group (1 mentions)
Ssea 1 pe, by BioLegend (1 mentions)
Facsaria sorter, by BD (1 mentions)
Cd49b pe, by BioLegend (1 mentions)
Sca 1 pe cy7, by BioLegend (1 mentions)
Streptavidin apc cy7, by BioLegend (1 mentions)
Cd31 biotin, by BioLegend (1 mentions)
Cd45 biotin, by BioLegend (1 mentions)
Ter119 biotin, by BioLegend (1 mentions)
Cd49f pe, by BioLegend (1 mentions)
Epcam apc, by Miltenyi Biotec (1 mentions)
Matrigel, by BD (1 mentions)
Cd45 pe cy7, by BioLegend (1 mentions)
Cd44 apc, by BD (1 mentions)
8 protocols using cd49f apc
1
Characterization of Dental and Bone Marrow Stem Cells
2
Mammary Gland Cell Sorting and Culture
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Thoracic and inguinal mammary glands were dissected from 9-week-old p38α(lox/lox);MMTV-Cre and MMTV-Cre virgin females or 6-week-old p38α(lox/lox);MMTV-Cre;PyMT and MMTV-Cre;PyMT female mice. Cell suspensions were prepared as previously described (Prater et al., 2013 (link)). FACS sorting was performed with a FACSAria sorter (BD Bioscience). Data were analyzed with the FlowJo software package. The antibodies used for FACS were from Miltenyi Biotec: Epcam-APC (#130102234), Epcam-FITC (#130102214), CD49f-APC (#130097250), CD49f-PE (#130097246), BP1-biotin (#130101844), CD31-biotin (#130101955), CD45-biotin (#130101952), and Ter119-biotin (#130101882); and from Biolegend: CD61-AF480 (#104311), Sca1-PECy7 (#108114), CD49b-PE (#103506), and Streptavidin-APCCy7 (#405208). FACS gating was based on single color staining and fluorescence-minus-one controls, and was checked with isotype controls (Prater et al., 2013 (link)). Gating strategies were as described previously (Prater et al., 2013 (link), Shehata et al., 2012 (link)). For colony-formation assays, freshly sorted cells were embedded in Matrigel (BD Pharmingen) as described previously (Shackleton et al., 2006 (link)).
3
Flow Cytometry Isolation of Mammary Cancer Cells
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MDA-MB-231, MCF7, and SKBR3 human cancer cell lines were trypsinized to single cells, and MMTV-NDL murine mammary tumors (1.0–1.5 cm in diameter) were harvested and dissociated to single cells as previously described [29 (link),30 (link)] with minor modifications. Cells were suspended at 1 × 107 per mL in staining buffer (PBS with 2% FBS) and incubated for 30 min on ice with antibodies. Cells were washed three times, re-suspended in staining buffer with 1 μg/mL Propidium Iodide (PI), analyzed, and sorted with a FACS Aria II cell sorter (Becton Dickinson, Franklin Lakes, NJ, USA). Sorting schemes were based on previously published studies for human cell lines [1 (link),8 (link)] and primary mouse tissues [33 (link),34 (link)]. Results were analyzed using FlowJo software. Antibodies used were CD24-PE-Cy7 (1:100 dilution, #561646) and CD44-APC (1:100 dilution, #559942; BD Pharmingen, San Diego, CA, USA) for human cells, and CD24-PE (1:200 dilution, #553262; BD Pharmingen), CD49f-APC (1:100 dilution, #313615; Biolegend, San Diego, CA, USA), CD31-PE-Cy7 (1:100, #102417; Biolegend), and CD45-PE-Cy7 (1:100, #103113; Biolegend) for mouse cells.
4
Immunophenotyping of Breast Cancer Cells
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The autologous breast cancer cells were stained with HER-2 PE, epithelial cell adhesion molecule (Ep-CAM) PE-Dazzle 594, mucin-1 (MUC-1) PE-Cy7 and integrin alpha 6 (CD49f) APC (Biolegend, USA) as recommended by the manufacturer. The cells were acquired on the LSRII flow cytometer and the data were analysed as indicated previously.
5
Single Cell Surface Marker Isolation
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Single cells were suspended in PBS, 2 mM EDTA, 0.5% BSA and stained with antibody for 15
min at 4°C. Fluorescence-activated cell sorting and analysis were performed on a BD Special
Order FACS Aria II system and Diva v6.1.1 (BD Biosciences). Live single cells were gated based on
scatter properties and analyzed for their surface marker expression as previously described
[15 (link)]. Antibodies used for FACS include Epcam-PE
(Miltenyi Biotech), CD44−FITC (ebioscience), and CD49f-APC (BioLegend).
min at 4°C. Fluorescence-activated cell sorting and analysis were performed on a BD Special
Order FACS Aria II system and Diva v6.1.1 (BD Biosciences). Live single cells were gated based on
scatter properties and analyzed for their surface marker expression as previously described
[15 (link)]. Antibodies used for FACS include Epcam-PE
(Miltenyi Biotech), CD44−FITC (ebioscience), and CD49f-APC (BioLegend).
6
Isolation and Characterization of Mammary Stem Cells
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MaSCs were isolated using mechanical and enzymatic digestion followed by fluorescence-activated cell sorting (FACS), as detailed previously (Asselin-Labat et al., 2006 (link), Sleeman et al., 2007 (link)). Cells were stained with either anti-CD24 Pacific Blue (catalog no. 582583; BD Biosciences) or EpCAM BV605 (catalog no. 563214; BD Biosciences), CD31 (catalog no. 561410)/CD45 (catalog no. 552848)/TER119 (catalog no. 557853) Pe Cy7 (BD Biosciences), Ly6A-PE (catalog no. 553108, BD Biosciences), and CD49f-APC (catalog no. 313616, Biolegend). Linear density contour plots were used to determine gates for stem cell populations. FMO staining control samples were used to determine the gates for SCA-1-negative cells. Flowlogic was used to analyze data and prepare figures.
7
Isolation of Murine Mammary Stem Cells
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Primary mammary epithelial cells (MECs) were prepared from 12-week-old FVB mice as previously described [13 (link)]. To separate MaSCs from the purified mouse MECs, cells were stained with CD24-FITC (BD Biosciences, San Jose, CA) and CD49f-APC (BioLegend, San Diego, CA), and CD24med/CD49fhigh cells was then collected using a FACSAria flow cytometer (BD Biosciences).
8
Isolation and Culture of Mammary Epithelial Cells
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Mouse mammary glands were dissected and analyzed as previously described16 (link). Briefly, the glands were digested with collagenase/hyaluronidase for 2 hours followed by ACK. Then a 2 minutes Trypsin and DNAse/Dispase digestion was performed. The cells were then stained with the following antibodies: EpCAM-FITC, CD49f-APC, CD24-PE-Cy7 and Linage-PacBlue (CD45, Ter119 and CD31) (Biolegend). Viable cells were identified based on forward- and side- scatter profiles and by DAPI exclusion. Single cells were analyzed and sorted using FACS Aria II (BD Bioscience). For in vitro colony forming assay, 10.000 sorted epithelial cells were plated into 96-well plate previously coated with 2.5% growth factor reduced matrigel. If not otherwise specified, irradiated L1-Wnt3a expressing cells were used as feeder layer. Cells were grown into liquid media supplemented with Y-27632 dihydrochloride (Sigma-Aldrich), murine recombinant EGF (Prepotech), rhR-Spondin1 (R&D) and Nogging (R&D), as previously described10 (link).
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