TPBG, also named as 5T4 and WAIF1, is an N-glycosylated transmembrane glycoprotein expressed in the cellsurface of many types of tumors including colorectal, ovarian, and gastric. TPBG is not only used as a prognostic marker in cancer but also is promising target of therapeutical antibody and cancer vaccine.
Split saturated culture 1:3-1:6 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
Cell Line Generation
293T mouse TPBG cell line was generated using a lentiviral vector expressing the mouse TPBG sequence.
Characterization using PCR sequencing and WB
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Application
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Cell Resuscitation
1. Prewarm culture medium (DMEM + 10% FBS + 1μg/mL Puromycin) in a 37°C water bath.
2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
7. Incubate the flask at 37°C, 5% CO2 incubator.
8. Split saturated culture 1:3-1:6 every 3 days; seed out at about 1-3 × 105 cells/mL.
Cell Freezing
1. Prepare the freezing medium (70% DMEM + 20% FBS + 10% DMSO) fresh immediately before use.
2. Keep the freezing medium on ice and label cryovials.
3. Transfer cells to a sterile, conical centrifuge tube, and count the cells.
4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
5. Resuspend the cells at a density of at least 3×106 cells/mL in chilled freezing medium.
6. Aliquot 1 mL of the cell suspension into each cryovial.
7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
8. Transfer vials to liquid nitrogen for long-term storage
References
1. Carsberg CJ, Myers KA, Evans GS, Allen TD, Stern PL (Aug 1995). "Metastasis-associated 5T4 oncofoetal antigen is concentrated at microvillus projections of the plasma membrane". Journal of Cell Science. 108.
2. King KW, Sheppard FC, Westwater C, Stern PL, Myers KA (Jun 1999). "Organisation of the mouse and human 5T4 oncofoetal leucine-rich glycoprotein genes and expression in foetal and adult murine tissues". Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression. 1445(3): 257–70.
