KC-4296

NCI-H1299-ROCK2-KO-3C3-Cell-Line

44228

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Background of NCI-H1299-ROCK2-KO-3C3-Cell-Line

Rho-associated protein kinase (ROCK), downstream effector of Rho GTPases, is a kind of serine-threonine protein kinase. Two mammalian ROCK homologs, ROCK1 and ROCK2, have been identified, which share 92% identity of amino acid sequence in their kinase domain and have a similar structure. In its native state, ROCK is self-suppressed by the pH domain in the carboxyl-terminal region. However, when RhoA and (or) RhoC are activated to bind to the RBD domain or the auto-inhibitory region is cleaved and removed by caspase-3 or Granzyme-B, ROCK is activated. As a well-described signaling cascade transmission, RhoA/ROCK pathway is an inhibitory cue to the growth cone by modulation of actin dynamics. Within this pathway, ROCK has a vital role on inhibitory signals for axon growth and in the regulation of cell survival. Although ROCK is ubiquitously expressed in all tissues, ROCK1 is mainly expressed in non-neuronal tissue and ROCK2 is expressed in brain and the spinal cord abundantly and improves with age.

Specifications

Catalog Number	KC-4296
Cell Line Name	NCI-H1299-ROCK2-KO-3C3-Cell-Line
Host Cell Line	NCI-H1299
Description	Stable NCI-H1299 cell line with human ROCK2 gene knockout, No.3C3
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% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640 + 10% FBS
Selection Marker	N/A
Morphology	Fibroblastoid cells growing as a monolayer
Subculture	Split the saturated culture at a ratio of 1:3~1:5 every 2~3 days; seed out at about 1-3 x 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

NCI-H1299-ROCK2-KO 3C3 cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of NCI-H1299-ROCK2-KO 3C3 cell line stable clone using PCR sequencing.

Figure 2: Characterization of NCI-H1299-ROCK2-KO 3C3 cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of NCI-H1299-ROCK2-KO 3C3 cell line stable clone using western blot.

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 10% FBS)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 the saturated culture at a ratio of 1:3 ~ 1:5 every 2~3 days; seed out at about 1-3 x 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI1640 + 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

Lu W, Wen J, Chen Z. Distinct Roles of ROCK1 and ROCK2 on the Cerebral Ischemia Injury and Subsequently Neurodegenerative Changes. Pharmacology. 2020;105(1-2):3-8. doi: 10.1159/000502914. Epub 2019 Sep 19. PMID: 31537002.

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.