KC-0147

HEK293-APP695-Swedish Cell Line

64073

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Background of HEK293-APP695-Swedish Cell Line

Amyloid precursor protein (APP) is a type I membrane protein with several human isoforms due to alternative splicing. APP770, 751, and 733 contain a Kunitz protease inhibitor (KPI) domain (residue 291-342) and APP695 does not. APP is a cell surface molecule with many functions. It can be processed proteolytically in two different pathways. In one pathway, beta - and gamma -secretase cleave at the beta site betweenresidue 670 and 671 and the gamma site between residue 711 and 714 to produce beta -amyloid peptide (A beta 40 and A beta 42), a major component in plaques found in brains of patients with Alzheimer's disease.The other pathway involves alpha -secretase that cleaves residues between 687 and 688. It is antiamyloidogenic due to its benign character and the prevention of the A beta peptide formation.

Specifications

Catalog Number	KC-0147
Cell Line Name	HEK293-APP695-Swedish Cell Line
Clone Number	NA
Host Cell Line	HEK293
Description	HEK293 cell line stable expressing exogenous human APP695 gene bearing Swedish mutation.
Quantity	One vial 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% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS +500ug/mL G418
Selection Marker	G418
Morphology	Fibroblastoid cells growing as monolayer
Subculture	Split saturated culture 1:4-1:5 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

HEK293-APP695 cell line was generated using pCDNA3.1 vector expressing human APP695 sequence bearing swedish mutation.

Characterization

Figure 1:Characterization of APP695 overexpressing in HEK293 stable clones using WB.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS and 500ug/mL G418)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 1:4-1:5 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. Mullan M, Crawford F, Axelman K, Houlden H, Lilius L, Winblad B, Lannfelt L (August 1992). "A pathogenic mutation for probable Alzheimer's disease in the APP gene at the N-terminus of beta-amyloid".
2. Wilson CA, Doms RW, Lee VM (August 1999). "Intracellular APP processing and A beta production in Alzheimer disease". Journal of Neuropathology and Experimental Neurology. 58 (8): 787–94.

Use License Agreement

Research Use Only.

Not for use in diagnostic procedures or therapeutic applications.

Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.