KC-6388

CHOK1-PEAR1 Cell Line

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Background of CHOK1-PEAR1 Cell Line

PEAR1, also known as platelet endothelial aggregation receptor 1, is known to play a crucial role in the migration and differentiation of muscle satellite cells (MuSCs). Platelet Endothelial Aggregation Receptor (PEAR1), as a platelet receptor, plays a vital role in hemostasis. This receptor, by its extracellular part, causes platelet adhesion and consequently initiates platelet aggregation. Dysfunction of PEAR1 can disrupt platelet aggregation in patients with cardiovascular diseases (CVDs). Diseases associated with PEAR1 include Bernard-Soulier Syndrome.

Specifications

Catalog Number	KC-6388
Cell Line Name	CHOK1-PEAR1 Cell Line
NCBI/UniProt Accession Number	NM_001080471.3
Clone Number	14#
Host Cell Line	CHOK1
Description	Stable CHOK1 clone expressing exogenous human PEAR1 gene
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 + 10μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 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 24 hours
Mycoplasma Status	Negative

Cell Line Generation

CHOK1-PEAR1 cell line was generated using a lentiviral vector expressing the human PEAR1 sequence.

Characterization

Figure 1: Characterization of human PEAR1 overexpression in the CHOK1-PEAR1 stable clone using FACS.

Figure 2: Characterization of human PEAR1 in the CHOK1 stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 + 10% FBS + 10μg/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 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% RPMI1640 + 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. Ansari N, Najafi S, Shahrabi S, Saki N. PEAR1 polymorphisms as a prognostic factor in hemostasis and cardiovascular diseases. J Thromb Thrombolysis. 2021 Jan;51(1):89-95. doi: 10.1007/s11239-020-02149-w. PMID: 32445063.
2. Zhao Y, Zhang L, Hao R, Li S, Li S, Shi S, Tong H, Liu B. PEAR1 Promotes Myoblast Proliferation Through Notch Signaling Pathway. Biology (Basel). 2024 Dec 19;13(12):1063. doi: 10.3390/biology13121063. PMID: 39765730; PMCID: PMC11673774.
3. Zhang S, Qu K, Lyu S, Hoyle DL, Smith C, Cheng L, Cheng T, Shen J, Wang ZZ. PEAR1 is a potential regulator of early hematopoiesis of human pluripotent stem cells. J Cell Physiol. 2023 Jan;238(1):179-194. doi: 10.1002/jcp.30924. Epub 2022 Nov 27. PMID: 36436185.