After MCF10DCIS cells reached ~80% confluence, they were trypsinized, filtered with a 40 μm cell strainer (Fisher Scientific), and incubated with CD44/FITC (BD Biosciences, Cat No. 555478), CD49f/APC (eBioscience, Cat No. 17-0495-80) or CD24/PE (BD Biosciences, Cat No. 555428) antibodies and 0.5 μg/ml propidium iodide (Sigma) at 4°C for 30 min. CD44+/CD49f+/CD24− cells that did not take up PI were then sorted into 96-well plates at 1 cell/well using 70 μm nozzle and incubated overnight at 37°C. On day one post-sorting, the plate was monitored under a bright-field microscope equipped with an X-Y stage to confirm that each well contained only one cell. The wells that contained more than one cell or no cells were excluded and the wells containing single cells were monitored everyday.
Cd49f apc
Manufactured by Thermo Fisher Scientific
CD49f-APC is a fluorescently-labeled monoclonal antibody that binds to the CD49f cell surface antigen, also known as alpha-6 integrin. It is commonly used in flow cytometry applications for the identification and analysis of cells expressing this cell surface marker.
Automatically generated - may contain errors
Lab products found in correlation
Cd44 fitc, by BD (2 mentions)
Cd24 pe, by BD (2 mentions)
Facsaria 3 cell sorter, by BD (2 mentions)
Cd71 pe, by BioLegend (2 mentions)
Ham s f 12, by Merck Group (2 mentions)
Cholera toxin, by Merck Group (2 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (2 mentions)
Facsdiva, by BD (2 mentions)
Cacl2, by Merck Group (2 mentions)
40 μm cell strainer, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Epcam pe, by Thermo Fisher Scientific (1 mentions)
Facsaria, by BD (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Ter119 pecy7, by Thermo Fisher Scientific (1 mentions)
Cd24 pe, by Thermo Fisher Scientific (1 mentions)
Facsverse, by BD (1 mentions)
Ter119 pe, by Thermo Fisher Scientific (1 mentions)
Epcam fitc, by Thermo Fisher Scientific (1 mentions)
6 protocols using cd49f apc
1
Isolation and Culture of MCF10DCIS Stem Cells
2
Characterizing Cell Surface Markers in DEAR1-KD Clones
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Control clones and DEAR1-KD 76N-E6 clones were grown in 2D culture and stained for FACS using EpCAM-PE (eBioscience, 12-9326-41), CD49f-APC (eBioscience, 17-0495-80), CD24-PE (BD Biosciences, 555428), and CD44-FITC (BD Biosciences, 555478) according to the manufacturer’s Staining Cell Surface Antigens for Flow Cytometry protocol. Plated cells were trypsinized, counted, and resuspended in Flow Cytometry Staining Buffer for a final concentration of 1 × 107 cells/mL. 50 µL of the cell suspension was incubated with primary antibody so that the final volume was 100 µL per sample. The cells incubated for 30 min in the dark at 4 °C, washed, and analyzed. For these experiments, 2 stable control vector (CshR) clones and 2 DEAR1-KD (DshR) stable clones were used experiments were performed in triplicate.
3
Primary KC Isolation and Culture
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A suspension of primary KCs with 5.0 × 106cells/ml was incubated with CD71-PE (1:200, BioLegend) and CD49f-APC (1:85, eBioscience). The selection of α6bri/CD71dim cells was performed using a FACSAriaIII Cell Sorter and FACSDiva software (BD Biosciences). Sorted α6bri/CD71dim cells were plated at 1.8 × 103 cells/cm2 onto the feeder layer and cultured in FAD medium [1-part Ham’s F-12 (Sigma-Aldrich) and 3-parts DMEM supplemented with 10% non-inactivated FBS, 1.8 × 104 M adenine, 0.5 μg/ml hydrocortisone, 5 μg/ml insulin, 10–10 M cholera toxin (Sigma-Aldrich), 10 ng/ml epidermal growth factor (Peprotech), 1.8 mM CaCl2 (Merck), and 1% PenStrep].
4
Labeling and Sorting Cell Populations
5
Isolation and Characterization of Mammary Epithelial Cells
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After freshly isolated mouse mammary gland fragments were cultured in PIC hydrogels or Matrigel for seven days, the generated MGOs were collected as described above. After washing with cold PBS twice, MGOs were digested in TrypLE for 15–20 min at 37 °C to obtain single‐cell suspensions of mammary epithelium. For flow cytometry and sorting, cells were stained with CD31‐PE/cy7 (Antibodychain, Cat. #1 112 040), CD45.2‐BV510 (Antibodychain, Cat. #1 149 185), TER119‐PE (eBioscience, Cat. #12‐5921‐81), CD45.2‐PerCP (BD, Cat. #552 950), EpCAM‐FITC (eBioscience, Cat. #11‐5791‐82), and CD49f‐APC (eBioscience, Cat. #17‐0495‐80). Flow cytometry analysis was conducted on BD FACS Verse and FACS sorting was performed on a FACS ARIA III. Fixable Viability Dyes (FVD) eFluor 450 was added to distinguish dead and live cells. Data were analyzed using FlowJo V10 software and FACS data are presented in dot plots with equal number of events. The gating strategy for the mammary epithelium is shown in Figure S10 (Supporting Information).
6
Primary KC Isolation and Culture
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A suspension of primary KCs with 5.0 × 106cells/ml was incubated with CD71-PE (1:200, BioLegend) and CD49f-APC (1:85, eBioscience). The selection of α6bri/CD71dim cells was performed using a FACSAriaIII Cell Sorter and FACSDiva software (BD Biosciences). Sorted α6bri/CD71dim cells were plated at 1.8 × 103 cells/cm2 onto the feeder layer and cultured in FAD medium [1-part Ham’s F-12 (Sigma-Aldrich) and 3-parts DMEM supplemented with 10% non-inactivated FBS, 1.8 × 104 M adenine, 0.5 μg/ml hydrocortisone, 5 μg/ml insulin, 10–10 M cholera toxin (Sigma-Aldrich), 10 ng/ml epidermal growth factor (Peprotech), 1.8 mM CaCl2 (Merck), and 1% PenStrep].
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