KC-5874

HT1080-ACP3-Middle Cell Line

59021

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Background of HT1080-ACP3-Middle Cell Line

ACP3, also known as TM-PAP. This gene encodes an enzyme that catalyzes the conversion of orthophosphoric monoester to alcohol and orthophosphate. It is synthesized under androgen regulation and is secreted by the epithelial cells of the prostate gland. An alternatively spliced transcript variant encoding a longer isoform has been found for this gene. This isoform contains a transmembrane domain and is localized in the plasma membrane-endosomal-lysosomal pathway. Research has shown that TMPAP is a novel therapeutic target in advanced prostate cancer.

Specifications

Catalog Number	KC-5874
Cell Line Name	HT1080-ACP3-Middle Cell Line
NCBI/UniProt Accession Number	NM_001134194.1
Clone Number	77#
Host Cell Line	HT1080
Description	Stable HT1080 cell line expressing exogenous ACP3 gene in middle level
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% RPMI 1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI 1640 + 10% FBS + 1μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:3-1:4 every 2-3 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

HT1080 ACP3 Cell Line was generated using a lentiviral vector expressing the ACP3 sequence.

Characterization

Figure 1: Characterization of ACP3 overexpression in the HT1080 ACP3 Middle stable clone using FACS.

Figure 2:Characterization of ACP3 overexpression in the HT1080 stable clone using DNA sequencing.

Cell Resuscitation

Prewarm culture medium (RPMI 1640 + 10% FBS + 1μg/mL Puromycin) 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 1:3-1:4 every 2-3 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

Kirschenbaum A, Cheung P, Rajagopalan V, Yao S, Milgrim L, Kyprianou N, Levine AC. Transmembrane prostatic acid phosphatase: a therapeutic target in advanced prostate cancer. Am J Clin Exp Urol. 2024 Oct 15;12(5):255-265.