KC-1404

T47D-PalR-Cell-Line

22073

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Background of T47D-PalR-Cell-Line

Palbociclib, branded as Ibrance, is a selective inhibitor of cyclin-dependent inhibitor of CDK4 and CDK6, and was approved for treatment of HR-positive and HER2 negative breast cancer.

Specifications

Catalog Number	KC-1404
Cell Line Name	T47D-PalR-Cell-Line
Host Cell Line	Human T47D cell line
Description	Stable T47D cell clone resistant to CDK4/6 inhibitors
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	70% RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10%FBS+0.2 Units/mL Insulin+5µM Palbociclib
Morphology	Epithelial
Subculture	Split saturated culture 1:3-1:5 every 3 days; seed out at about 1-3 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

T47D PALR cell Line was generated after long time Palbociclib treatment.

Characterization

Figure 1. Characterization of CDK pathway in T47D-PalR stable clone using Western Blot and RNAseq.

Figure 2. kinase inhibitors screening.

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 10% FBS + 0.2Units/mL Insulin + 5µM Palbociclib)in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split saturated culture 1:3-1:5 every 3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI-1640 + 20% FBS + 10% DMSO) fresh immediately before use.
Keep the freezing medium on ice and label cryovials.
Transfer cells to a sterile, conical centrifuge tube, and count the cells.
Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
Aliquot 1 mL of the cell suspension into each cryovial.
Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
Transfer vials to liquid nitrogen for long-term storage

References

McCartney, Amelia; Migliaccio, Ilenia; Bonechi, Martina; Biagioni, Chiara; Romagnoli, Dario; De Luca, Francesca; Galardi, Francesca; Risi, Emanuela; De Santo, Irene; Benelli, Matteo; Malorni, Luca; Di Leo, Angelo (23 July 2019). "Mechanisms of Resistance to CDK4/6 Inhibitors: Potential Implications and Biomarkers for Clinical Practice". Frontiers in Oncology