Immortalized Human Mammary Epithelial Progenitor (K5+/K19+) Cells - hTERT

CAT.NOUNITPRICE
T04521x106 cells / 1.0 ml
$0.00

Specifications


Description

The immortalized human mammary progenitor cells – hTERT (K5+/K19+) is a clonal cell population of progenitors coexpressing normal mammary and stem cell markers. It has the ability to self-renew and differentiate into luminal and/or myoepithelial cell lineages. Through propagation in different optimized media, the cells are able to give rise to new population of cells with different morphology and characteristics, such as mucin-1 positive cells, vimentin-negative cells, or expressing basal, luminal, and other stem cell markers. Aldehyde dehydrogenase 1A3 enzyme is noticeably higher in expression, a marker commonly used for isolating normal and tumor mammary stem cells. K5+/K19+ cells have Wnt, Notch, and hedeghog gene regulatory pathways. It is a valuable tool in research in the field of biology, the stem/progenitor origin, and the heterogeneity mechanisms in breast cancer.

SKUT0452
SpeciesHuman (H. sapiens)
Tissue/Organ/Organ SystemBreast
Growth PropertiesAdherent
Cell MorphologyCobble-stone|Spindle shaped
Immortalization MethodSerial passaging and transduction with recombinant retroviruses carrying hTERT gene
Applications

For Research Use Only

Mammalian SelectionNeomycin
Unit quantity1x106 cells / 1.0 ml
Cell TypeImmortalized Cells
Expression Profile

K5, K14, vimentin, E-cadherin, p63, K8, K18, K19, CD29, CD49f
a-SMA, CD10, Thy-1 when grown in DCFI-1

Propagation Requirements

Use of PriCoatTM T25 Flasks (G299) or Applied Cell Extracellular Matrix (G422) is required for cell adhesion to the culture vessels. Grow cells in ECM-coated culture vessels unless otherwise specified in the Propagation Requirements below.

Optimized Media:
To express basal, luminal, and stem cell markers with some myoepithelial cell markers (a-SMA, CD10, Thy-1), grow cells in DFCI-1 media.
To make DFCI-1 the base medium is Prigrow VIII and Prigrow IV (1:1, vol/vol) medium available at abm (TM018 and TM004). To make the complete growth medium, add the following components to the base medium: fetal bovine serum (TM999)* to a final concentration of 1%, Penicillin/Streptomycin Solution (G255) to a final concentration of 1%, HEPES (Gibco) to a final concentration of 10 mM, 12.5 ng/ml Recombinant Human EGF (Z100135) to a final concentration of 12.5 ng/ml, 6.5 ng/ml triiodothyronine (Sigma), 0.545 ng/ml beta-estradiol (Sigma), 1 µg/ml insulin (TM053), 1 µg/ml hydrocortisone (Sigma), 0.006X ethanolamine (Sigma), 14.1 µg/ml phosphoethanolamine (Sigma), 10 µg/ml transferrin (Sigma), 2 mM L-glutamine (G275), 2.6 ng/ml sodium selenite (Sigma), 1 ng/ml cholera toxin (Sigma), 35 µg/ml bovine pituitary extract (Hammond Cell Tech), and 10 µg/ml ascorbic acid (Sigma). Adjust pH to 7.4.
Change media every 2-3 days.
Carbon dioxide (CO2): 5%, Temperature: 37.0°C.
* Do not use heat-inactivated FBS for cell culture unless specified otherwise.

For cells to adopt spindle-shape morphology around tight epithelial cell colonies and for the presence of mucin-1 positive cells: grow cells in mammary epithelial growth medium (MEGM; Lonza) supplemented with B27 (10ml/500ml medium), 20 ng/ml Recombinant Human EGF (Z100135), 20 ng/ml Recombinant Human FGF2 (Z101455), 4 µg/ml heparin, and Penicillin/Streptomycin Solution (G255) to a final concentration of 1%.
Carbon dioxide (CO2): 6.5%, Temperature: 37.0°C.

For luminal differentiation, the absence of myoepithelial differentiation, and the appearance of mucin-1 positive and vimentin-negative cells: grow the cells in DCFI-2 media where the base is Prigrow VIII and Prigrow IV (1:1, vol/vol) medium available at abm (TM018 and TM004). Supplement with 10 mM HEPES (Gibco), 12.5 ng/ml Recombinant Human EGF (Z100135), 6.5 ng/ml triiodothyronine (Sigma), 0.545 ng/ml beta-estradiol (Sigma), 1 µg/ml insulin (TM053), 1 µg/ml hydrocortisone (Sigma), 0.006X ethanolamine (Sigma), 14.1 µg/ml phosphoethanolamine (Sigma), 10 µg/ml transferrin (Sigma), 2 mM L-glutamine (G275), 2.6 ng/ml sodium selenite (Sigma), 1 ng/mg cholera toxin (Sigma), 0.05% bovine serum albumin (BSA), Penicillin/Streptomycin Solution (G255) to a final concentration of 1%, and 10 µg/ml ascorbic acid (Sigma). Adjust pH to 7.4.
Carbon dioxide (CO2): 6.5%, Temperature: 37.0°C.

Preservation Protocol1. Freeze Medium: Complete growth medium with 20% FBS and 10% DMSO.
2. Storage Temperature: Liquid nitrogen vapour phase.
QC

1) Western blot to assess cell lineage-related and stem cell markers; 2) Immunofluorescence staining analysis for lineage markers; 3) Gene expression profiling for gene regulatory pathways

Disclaimer

1. For for-profit organizations and corporations, please contact [email protected] for pricing of this item.

2. Sale of this item is subjected to the completion of a Material Transfer Agreement (MTA) by the purchasing individual/institution for each order. If you have any questions regarding this, please contact us at [email protected].

3. All test parameters provided in the CoA are conducted using abm's standardized culture system and procedures. The stated values may vary under the end-user's culture conditions. Please verify that the product is suitable for your studies by referencing published papers or ordering RNA (0.5 μg, Cat.# C207, $450.00) or cell lysate (100 μg, Cat.# C206, $600.00) to perform preliminary experiments, or alternatively use our Gene Expression Assay Service (Cat# C138). All sales are final.

4. We recommend live cell shipments for ease of cell transfer and this option can be requested at the time of ordering. Please note that the end-user will need to evaluate the feasibility of live cell shipment by taking into account the final destination's temperature variation and its geographical location. In addition, we thoroughly test our cell lines for freeze-thaw recovery. If frozen cells were received and not recovered in your lab under the exact, specified conditions (using recommended culture vessel, media, additional supplements, and atmospheric conditions), a live cell replacement is possible at a cost (plus shipping).

5. All of abm's cell biology products are for research use ONLY and NOT for therapeutic/diagnostic applications. abm is not liable for any repercussions arising from the use of its cell biology product(s) in therapeutic/diagnostic application(s). Please contact a technical service representative for more information.

6. abm makes no warranties or representations as to the accuracy of the information on this site. Citations from literature and provided for informational purposes only. abm does not warrant that such information has been shown to be accurate.

7. abm warrants that cell lines shall be viable upon initiation of culture for a period of thirty (30) days after shipment and that they shall meet the specifications on the applicable abm Material Product Information sheet, certificate of analysis, and/or catalog description. Such thirty (30) day period is referred to herein as the "Warranty Period."

DepositorUniversity of Nebraska Medical Center
FAQs


I want to make sure these cells express my gene of interest before I decide to buy the cell line. Can you provide a sample so this can be tested?
We do not carry out downstream characterization or gene expression profiling of our cell lines. To facilitate your preliminary experiments we can provide an RNA extraction (0.5ug total RNA) or cell lysate (100ug/100ul provided in 62.5mM Tris‐HCl, 2% SDS, 10% Glycerol, 50mM DTT, 0.01% w/v Bromophenol Blue) for any of our immortalized cell lines for a small fee. Please inquire directly for more information. The lead time will be around 2 weeks from the time of placing an order (if the item is in stock).
How often do I need to change the media?
The media should be changed every 2-3 days.
Why do these cells need bio safety level II?
In order to be more cautious, we follow the CDC-NIH recommendations that all mammalian sourced products should be handled at the Biological Safety Level 2 to minimize exposure of potentially infectious products. This information can be found in 'Biosafety in Microbiological and Biomedical Laboratories' (1999). Your institution's Safety Officer or Technical Services will be able to make the call as to whether BioSafety Level I is possible with these cells at your site if required.
Do you sell ECM coated T75 flasks?
Yes we can provide a coating service. Please inquire with [email protected]
What can I coat a larger dish to subculture?
We also offer applied extracellular matrix (collagen type I) in liquid form, for the coating of larger flasks and other required plasticware: http://www.abmgood.com/Applied-Cell-Extracellular-Matrix-G422.html
How long can I store frozen vials for?
Cells that are properly frozen using an effective cryoprotective agent can be stored in liquid nitrogen indefinitely without affecting their recovery.
Should the cap of the flask be changed before starting the cell culturing step?
No, there is no need in sterile biosafety cabinets unless it has contacted any non-sterile condition (e.g. touching the contaminated tip, etc.).
What is the recommended storage temperature?
In general, if you received: Live cells: acclimatize for 3-4 hrs at at the recommended conditions stated for the cell line under the propagation section, and then change media afterwards. Frozen cells: Immediately place cells in liquid nitrogen; -180C.
How is cell density crucial for drug selection?
If antibiotic selection is applicable to the target cells, we suggest getting rid of all the background cells so that the cell density is kept lower (even 20-30%). However, once the clones are selected by clonal dilution, we don't need the drug to still be present. If needed, the cell density should be towards the higher end since cells are already selected. Any primary cells still present will be depleted as a result of senescence and the cell population that remains will be resistant to the specific antibiotic.
My cells are not detaching, what method do you recommend to trypsinize the cells?
1. Incubate the coated plate containing trypsin solution at recommended temperature indicated in the propagation section for 3-5 min till the cells round up, monitoring from time to time under microscope. 2. Diluting G422 (1:1) with PBS and coating for lesser time. Sometimes the collagen content in G422 is higher and thus make stronger bonding with cells. 3. You can try reducing the incubation time as well for coating the plate to make a thinner layer.
Why is it important to determine the optimal seeding density?
The seeding density we recommend is for when cells are plated to a new vessel. The optimal seeding density should allow cells to attach to the surface and have room to proliferate. If you seed too little, cells may not attach well to the surface (for adherent cells). Seeding density is important as many cells (adherent or suspension cells) need to be in close proximity for better growth. Cell-cell interactions allow cells to communicate with each other in response to changes in their microenvironment. This ability to send and receive signals is essential for the survival of the cell. In other cases, if the seeding density is too low, cells may attach but a retardation in cell growth is observed. If you seed too high, the cells will attach but there is insufficient room for further proliferation and they will stop replicating.
References


15
  • Zhao, X et al. "Telomerase-immortalized human mammary stem/progenitor cells with ability to self-renew and differentiate." Proc Natl Acad Sci USA 107(32):14146-14151 (2010). DOI: 10.1073/pnas.1009030107.
  • Zhao, X et al. "Derivation of myoepithelial progenitor cells from bipotent mammary stem/progenitor cells." PLoS One 7(4):e35338 (2012). DOI: 10.1371/journal.pone.0035338.
  • Band, V et al. "Distinctive traits of normal and tumor-derived human mammary epithelial cells expressed in a medium that supports long-term growth of both cell types" Proc Natl Acad Sci USA 86(4):1249-1253 (1989). PubMed: 2919173.
  • Zhao, X et al. "A block in lineage differentiation of immortal human mammary stem / progenitor cells by ectopically-expressed oncogenes." J Carcinog 10:39 (2011). DOI: 10.4103/1477-3163.91415.
  • Paranjape, AN et al. "Introduction of SV40ER and hTERT into mammospheres generates breast cancer cells with stem cell properties" Oncogene 31(15):1896-1909 (2012). DOI: 10.1038/onc.2011.378.
  • Dimri, G et al. "Mammary epithelial cell transformation: insights from cell culture and mouse models" Breast Cancer Res 7(4):171-179 (2005). PubMed: 15987472.