Py230

The Py230 cell line is an epithelial-like murine mammary luminal tumor cell line that has properties similar to those of normal mouse mammary stem cells. It was derived from a mammary adenocarcinoma that spontaneously arose in a MMTV-PyMY (mouse mammary tumor virus promoter driven Polyoma middle T-antigen) transgenic C57BL/6 female mouse. This cell line carries the Polyoma virus middle T oncogene. (L Ellies, personal communication)

Py230 cells are cuboidal-shaped and grow in well-differentiated colonies at confluence in culture. (PubMed:  24368187)

This cell line forms luminal tumors and lung metastasis in vivo. (Deposit Form)

Py230 cells differentiate into single positive luminal, myoepithelial, and alveolar cells upon stimulation with lactogenic hormones. (L Ellies, personal communication)

Py230 cells express luminal-epithelial markers E-cadherin, cytokeratin 8 and cytokeratin14. Py230 cells are negative for expression of estrogen receptor, progesterone receptor and express low levels of HER2. (PubMed:  24368187) 

The Py230 cell line has a more differentiated phenotype and grows less aggressively in cell culture in comparison to the undifferenitiated and mesenchymal CRL-3280, Py8119 cells, which were also derived from a MMTV-PyMY transgenic C57BL/6 female mouse. (PubMed:  22531600)

Py230 cells, along with Py8119 cells, are a pair of murine mammary cell lines with distinct epithelial-like (Py230) or mesenchymal (Py8119) features derived from MMTV-PyMT transgene-induced mammary tumors in C57BL/6 mice that can be useful to study mammary tumorigenesis. (PubMed:  24368187) 

1. Check all containers for leakage or breakage.
2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.

• Fetal bovine serum (FBS; ATCC 30-2020) for a final concentration of 5%
• MITO+ Serum Extender (Corning® #355006) for a final concentration of 0.1%

To insure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at -70°C.  Storage at -70°C will result in loss of viability.  

1. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water.  Thawing should be rapid (approximately 2 minutes).
2. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
3. Transfer the vial contents to a centrifuge tube containing  9.0 mL complete culture medium. and spin at approximately 125 x g for 5 to 7 minutes.
4. Resuspend cell pellet with the recommended complete medium (see the specific batch information for the culture recommended dilution ratio). It is important to avoid excessive alkalinity of the medium during recovery of the cells.  It is suggested that, prior to the addition of the vial contents, the culture vessel containing the complete growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6). pH (7.0 to 7.6).
5. Incubate the culture at 37°C in a suitable incubator.  A 5% CO₂ in air atmosphere is recommended if using the medium described on this product sheet.

Note: these cells will not maintain their differentiation properties without MITO+ Serum Extender.

Note: these cells grow slowly

Volumes used in this protocol are for 75 cm² flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.

1. Remove and discard culture medium. Briefly rinse the cell layer with Ca++/Mg++ free Dulbecco's phosphate-buffered saline (D-PBS) (ATCC 30-2200) or 0.25% Trypsin – 2.21mM EDTA in HBSS (Corning cat no. 25-053-Cl) solution to remove all traces of serum which contains trypsin inhibitor.

2. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).

    Note:  These cells will form small clumps upon trypsinization.They do not form a single cell suspension. To avoid clumping, do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.

3. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting. An inoculum of 3 X 10⁴ to 5 X 10⁴ viable cells/cm² is recommended.

4. Add appropriate aliquots of the cell suspension to new culture vessels. Incubate cultures at 37°C. Subculture when cell density reaches between 2 X 10⁵ and 3 X 10⁵ cells/cm².

Subcultivation Ratio: 1:3 to 1:6 is recommended.

Medium renewal: every other day

Note: These cells will differentiate if maintained at low density.

The product is provided 'AS IS' and the viability of ATCC^® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

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