Easysep mouse mammary stem cell enrichment kit

Manufactured by STEMCELL

Sourced in Canada

The EasySep Mouse Mammary Stem Cell Enrichment kit is a laboratory tool designed to isolate and enrich mouse mammary stem cells from a heterogeneous cell population. It utilizes antibody-mediated positive selection to target and separate the desired cell type.

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Lab products found in correlation

Pacific blue anti mouse cd24, by BioLegend (2 mentions) Lsrfortessa, by BD (2 mentions) Aria 2, by BD (2 mentions) Anti mouse cd29 apc, by BioLegend (2 mentions) Collagenase hyaluronidase solution, by STEMCELL (1 mentions) Gallios flow cytometer, by Beckman Coulter (1 mentions) Anti cd49f fitc, by STEMCELL (1 mentions) Dnase 1, by Merck Group (1 mentions) Hyaluronidase, by Merck Group (1 mentions) Collagenase, by Merck Group (1 mentions) Dispase 2, by Merck Group (1 mentions) Epicult b medium, by STEMCELL (1 mentions) 40 μm cell strainer, by BD (1 mentions) Facsaria, by BD (1 mentions) 7 aminoactinomycin d 7 aad, by BD (1 mentions)

Spelling variants of this product

EasySep kits (21 citations) Stem Cell Kits (3 citations)

5 protocols using easysep mouse mammary stem cell enrichment kit

Enrichment and Analysis of Mammary Stem Cells

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Lineage-positive cells were depleted from single-cell preparations using the EasySep Mouse Mammary Stem Cell Enrichment kit (#19757; Stemcell Technologies, Vancouver, BC, Canada). For transduced tumors, tdTomato fluorescence was used to positively select Lin− tumor cells for analysis and sorting. Single-cell preparations were resuspended (10⁷ cells/ml in HBSS+ (containing 2% fetal bovine serum with 10 mm HEPES buffer)) for antibody staining. Antibody incubations were performed on ice for 30 min. Cells were stained with anti-mouse CD24-Pacific Blue and anti-mouse CD29-APC (1:100; BioLegend, San Diego, CA, USA). Cells were either fluorescence-activated cell sorting analyzed (LSR Fortessa; BD Biosciences, Franklin Lakes, NJ, USA) or sorted (Aria II; BD Biosciences). Data analysis was performed using FlowJo, version 9.5.3 (Tree Star, Ashland, OR, USA).

Roarty K., Pfefferle A.D., Creighton C.J., Perou C.M, & Rosen J.M. (2017). Ror2-mediated alternative Wnt signaling regulates cell fate and adhesion during mammary tumor progression. Oncogene, 36(43), 5958-5968.

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Mammary Stem Cell Enrichment and Characterization

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Mouse mammary tissues were dissociated using Collagenase/Hyaluronidase solution (StemCell Technologies) and single cell suspensions were generated according to manufacturer's instructions. Lineage-depleted mammary epithelial cells were prepared using an EasySep mouse mammary stem cell enrichment kit (StemCell Technologies). Flow cytometry was performed by standard procedures using anti-mouse CD24-PE and anti-CD49f-FITC (StemCell Technologies). Fluorescence was recorded using Gallios Flow Cytometer (Beckman Coulter) and analyzed with Kaluza flow cytometry analysis software. Mammary tissues from three animals per genotype were analyzed with similar results. Percentages of stem cell-enriched cell populations in each genotype were quantitated. Lineage-depleted mammary epithelial cells were cultured for mammosphere formation for 7 days as previously described [30] (link).

Chung W.C., Zhang S., Challagundla L., Zhou Y, & Xu K. (2017). Lunatic Fringe and p53 Cooperatively Suppress Mesenchymal Stem-Like Breast Cancer. Neoplasia (New York, N.Y.), 19(11), 885-895.

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Mammary Stem Cell Enrichment and Profiling

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Lineage-positive cells were depleted from single cell preparations using the EasySep Mouse Mammary Stem Cell Enrichment kit (#19757; STEMCELL Technologies). For transduced tumors, tdTomato fluorescence was used to positively select Lin- tumor cells for analysis and sorting. Single cell preparations were resuspended (10⁷ cells/ml in HBSS+ (containing 2% FBS with 10mM HEPES buffer)) for antibody staining. Antibody incubations were performed on ice for 30 min. Cells were stained with anti-mouse CD24-Pacific Blue and anti-mouse CD29-APC (1:100; BioLegend). Cells were either FACS analyzed (LSR Fortessa; BD) or sorted (Aria II; BD). Data analysis was performed using FlowJo, version 9.5.3 (Tree Star).

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Isolation and Culture of Murine Mammary Epithelial Cells

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Mammary glands from 8 to 12-week-old virgin female mice were enzymatically digested and single cell suspensions of purified mammary epithelial cells (MECs) were obtained following a standard protocol^{49 (link)}. Briefly, mammary glands were digested at 37 °C for 1–2 h in Epi-Cult-B medium (Stem Cell Technologies Inc) with 600 U/ml collagenase (Sigma Aldrich) and 200 U/ml hyaluronidase (Sigma Aldrich). After lysis of the red blood cells with NH₄Cl, the remaining cells were washed with PBS/0.02% w/v EDTA. Cells were then dissociated with 0.25% w/v trypsin, 0.2% w/v EDTA for 2 min by gentle pipetting, then incubated in 5 mg/ml Dispase II (Sigma Aldrich) plus 1 μg/ml DNase I (Sigma Aldrich) for 5 min, followed by filtration through a 40 μM cell strainer (BD Falcon). MECs were then purified using the EasySep Mouse Mammary Stem Cell Enrichment Kit (Stem Cell Technologies Inc). MECs were seeded on top of 50 or 0.5 kPa Easy Coat hydrogels (Cell guidance system) coated with 10 μg/ml fibronectin and harvested after 24 h.

Bertolio R., Napoletano F., Mano M., Maurer-Stroh S., Fantuz M., Zannini A., Bicciato S., Sorrentino G, & Del Sal G. (2019). Sterol regulatory element binding protein 1 couples mechanical cues and lipid metabolism. Nature Communications, 10, 1326.

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Isolation and Characterization of Mammary Epithelial Cell Populations

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Mammary epithelial cells (MECs) were isolated using a standard protocol and non-epithelial cells were removed using the EasySep^™ Mouse Mammary Stem Cell Enrichment Kit (STEMCELL TECHNOLOGY, #19757). MECs were stained with biotinylated anti-CD24-fluorescein isothiocyanate (FITC, BD Biosciences), anti-CD49f-R-phycoerythrin (R-PE, BD Biosciences) and 7-amino-actinomycin D (7AAD, BD Biosciences). FACS analysis was performed using FACSAria (BD Biosciences). Only live cells were gated from the 7AAD negative portion and FACS analysis of the CD49f and CD24 cell population in mammary epithelial cells yielded four different cell populations: negative (CD24⁻CD49f⁻), luminal (CD24^hiCD49f^lo) and basal (CD24⁺CD49f^hi) cells. The latter ones were divided into two different populations: myoepithelial (CD24^loCD49f^hi) and the stem cell-enriched (CD24^midCD49f^hi). Our analysis and interpretation of the data are based on defined epitope distribution used by others (Shackleton et al., 2006 (link); Stingl et al., 2006 (link)) and our lab (Yamaji et al., 2009 (link)).

Yoo K.H., Kang K., Feuermann Y., Jang S.J., Robinson G.W, & Hennighausen L. (2014). The STAT5-regulated miR-193b locus restrains mammary stem and progenitor cell activity and alveolar differentiation. Developmental biology, 395(2), 245-254.

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