KC-4240

A549-ROCK2-KO-1B2-Cell-Line

44158

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Background of A549-ROCK2-KO-1B2-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-4240
Cell Line Name	A549-ROCK2-KO-1B2-Cell-Line
Host Cell Line	A549
Description	Stable A549 cell line with human ROCK2 gene knockout, No.1B2
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% McCoy's 5a + 20% FBS + 10% DMSO
Propagation Medium	McCoy's 5a + 10% FBS
Selection Marker	N/A
Morphology	Fibroblastoid cells growing as a monolayer
Subculture	Split the saturated culture at a ratio of 1:3~1:6 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

A549-ROCK2-KO 1B2 cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of A549-ROCK2-KO 1B2 cell line stable clone using PCR sequencing.

Figure 2: Characterization of A549-ROCK2-KO 1B2 cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of A549-ROCK2-KO 1B2 cell line stable clone using western blot.

Cell Resuscitation

Prewarm culture medium (McCoy's 5a + 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 ~ 1:6 every 2~3 days; seed out at about 1-3 x 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% McCoy's 5a + 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.