ROCK2 Knockout LL29 Cell Pool
Product classification
Welcome to consult Elem's high-throughput CRISPR service.
Contact us to orderROCK2 Knockout LL29 Cell Pool
Product ID： LM01122134318
Market price
Stock surplus
0
Antibody custom service consulting
隐藏域元素占位
- 产品描述
- 细胞复苏
- 细胞传代
- 细胞冻存
- 抗体验证结果
-
- Brand: ELEM粒曼
- Commodity name: ROCK2 Knockout LL29 Cell Pool
- Commodity ID: LM01122134318
- Gene Symbol: ROCK2 KIAA0619
- Ensembl ID: ENSG00000134318
- Uniprot ID: O75116
- 宿主细胞 / 类型: LL29/human lung fibroblast cell line
- NCBI Gene ID: 9475
- 规格: 1×10^6 cells/frozen vial
- 生长培养基: DMEM + 10% FBS + 1% double antibiotic solution
- 筛选标记: N/A
- 生长特性: Adherent cells, epithelial-like
- 培养条件: Incubator at 37°C with 5% CO2; passage at 1/2 to 1/3 confluence.
- 倍增时间: ~42 hours
- 参考换液频率: 2–3 times per week
- 支原体检测结果: Negative
- 敲除效率（Sanger测序）: 100%
- 蛋白质组验证结果: N/A
- 抗体货号: Adding...
- 目标基因介绍: Protein kinase which is a key regulator of actin cytoskeleton and cell polarity. Involved in regulation of smooth muscle contraction, actin cytoskeleton organization, stress fiber and focal adhesion formation, neurite retraction, cell adhesion and motility via phosphorylation of ADD1, BRCA2, CNN1, EZR, DPYSL2, EP300, MSN, MYL9/MLC2, NPM1, RDX, PPP1R12A and VIM. Phosphorylates SORL1 and IRF4. Acts as a negative regulator of VEGF-induced angiogenic endothelial cell activation. Positively regulates the activation of p42/MAPK1-p44/MAPK3 and of p90RSK/RPS6KA1 during myogenic differentiation. Plays an important role in the timely initiation of centrosome duplication. Inhibits keratinocyte terminal differentiation. May regulate closure of the eyelids and ventral body wall through organization of actomyosin bundles. Plays a critical role in the regulation of spine and synaptic properties in the hippocampus. Plays an important role in generating the circadian rhythm of the aortic myofilament Ca(2+) sensitivity and vascular contractility by modulating the myosin light chain phosphorylation.
- 细胞开发路径: A stable KO cell pool was generated using the CRISPR‑RNP approach; Sanger sequencing confirmed 100% knockout efficiency in the cell pool.
- 应用: A gene knockout cell pool with high knockout efficiency (KO Cell Pool) is particularly well suited for preliminary functional analyses, the development of complex disease models, precision drug screening, and broad‑scale genetic discovery studies. The KO pool can be directly applied to a variety of assays and analyses without the need for time‑consuming single‑clone selection, significantly enhancing experimental throughput.
Key words:- ROCK2 KIAA0619
-
01. Preheat the complete culture medium in a 37°C water bath.
02. Thaw the cryovial in a 37°C water bath for 1–2 minutes.
03. Transfer the cryovials into a biosafety cabinet and wipe their surfaces with 70% ethanol.
04. Unscrew the cap of the cryovial and gently transfer the cell suspension into a sterile centrifuge tube containing 9 mL of complete culture medium.
05. Centrifuge at room temperature at 125g for 5–7 minutes, then discard the supernatant.
06. Resuspend the cell pellet in 5 mL of complete culture medium, and transfer the cell suspension to a T25 culture flask.
07. Transfer the cells to a 37°C incubator with 5% CO2 for culture.
08. Recommended passage ratio: 1/2 to 1/3; cells reach confluence in 2–3 days. -
01. When the cell confluence in the culture flask reaches 80%–90% or higher, cells can be passaged.
02. Remove the culture medium, PBS, and trypsin (0.25% Trypsin‑EDTA, Gibco 25200‑056) from the 4°C refrigerator, place them in a 37°C water bath, and once their temperature approaches 37°C, take them out. Spray the exterior of the bottles with 75% ethanol, then transfer them to a biosafety cabinet.03. Remove the culture flask to be passaged from the incubator, spray the exterior of the flask with 75% ethanol, and place it inside a biosafety cabinet.
04. To avoid dispersing the cells, gently rinse the cells along the inner wall of the culture flask with PBS; discard the wash solution, and add 2 mL of PBS to the T25 flask.
05. Add the appropriate volume of trypsin (1.5 mL for a T75 flask, 0.5 mL for a T25 flask), and gently swirl the flask to ensure the enzyme evenly covers the cell‑covered bottom. Adjust the volume as needed based on the specific situation. After approximately 1–2 minutes, when most of the cells have detached, add the corresponding volume of complete culture medium to stop the digestion, then use a 5‑mL pipette to gently pipette up and down until all cells are fully detached.
06. Transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 300 × g for 5 minutes, and discard the supernatant.
07. Transfer 5 mL of complete culture medium to resuspend the cells, adjust the seeding ratio as needed, and replenish the culture flask with complete medium—add up to 13–15 mL for a T75 flask or 5 mL for a T25 flask—and supplement with 1% double antibiotic solution.
08. After capping the bottle and tightening the lid, gently shake the vial to ensure uniform cell suspension, then place it in a 37°C incubator with 5% CO2. -
01. Prepare the cryopreservation solution and pre-cool it in advance.
02. Ensure that the cells to be cryopreserved meet the cryopreservation requirements, and verify the following conditions under a microscope: healthy appearance and morphological characteristics, being in the late logarithmic growth phase, and showing no signs of contamination or senescence.
03. Digest and centrifuge the cells (refer to the subculture protocol for detailed steps).
04. Resuspend the cells in cryopreservation medium at 1 mL per tube, gently pipette up and down to ensure uniformity, and then aliquot into cryovials.
05. Place the cells in a programmed cooling box and freeze them in a −80°C freezer.
06. Subsequently, the cells are transferred to a liquid nitrogen tank for long-term storage. - In antibody validation
Previous Page
Next Page
Classification： Gene Knockout Cell Pool（KO Pool）
Cell Line Information
Gene Symbol
ROCK2 KIAA0619
NCBI Gene ID
9475
Ensembl ID
ENSG00000134318
Uniprot ID
O75116
Screening marker
N/A
Host cell/type
LL29/human lung fibroblast cell line
Specifications
1×10^6 cells/frozen vial
Growth Medium
DMEM + 10% FBS + 1% double antibiotic solution
growth characteristics
Adherent cells, epithelial-like
culture condition
Incubator at 37°C with 5% CO2; passage at 1/2 to 1/3 confluence.
doubling time
~42 hours
Reference fluid change frequency
2–3 times per week
Mycoplasma test results
Negative
Knock-out validation
Knockout efficiency (Sanger sequencing)
100%
Proteome Validation Results
N/A
Antibody number
Adding...
Antibody validation results
Cell Line Description
Introduction of target gene
Protein kinase which is a key regulator of actin cytoskeleton and cell polarity. Involved in regulation of smooth muscle contraction, actin cytoskeleton organization, stress fiber and focal adhesion formation, neurite retraction, cell adhesion and motility via phosphorylation of ADD1, BRCA2, CNN1, EZR, DPYSL2, EP300, MSN, MYL9/MLC2, NPM1, RDX, PPP1R12A and VIM. Phosphorylates SORL1 and IRF4. Acts as a negative regulator of VEGF-induced angiogenic endothelial cell activation. Positively regulates the activation of p42/MAPK1-p44/MAPK3 and of p90RSK/RPS6KA1 during myogenic differentiation. Plays an important role in the timely initiation of centrosome duplication. Inhibits keratinocyte terminal differentiation. May regulate closure of the eyelids and ventral body wall through organization of actomyosin bundles. Plays a critical role in the regulation of spine and synaptic properties in the hippocampus. Plays an important role in generating the circadian rhythm of the aortic myofilament Ca(2+) sensitivity and vascular contractility by modulating the myosin light chain phosphorylation.
Cell development path
A stable KO cell pool was generated using the CRISPR‑RNP approach; Sanger sequencing confirmed 100% knockout efficiency in the cell pool.
Application
A gene knockout cell pool with high knockout efficiency (KO Cell Pool) is particularly well suited for preliminary functional analyses, the development of complex disease models, precision drug screening, and broad‑scale genetic discovery studies. The KO pool can be directly applied to a variety of assays and analyses without the need for time‑consuming single‑clone selection, significantly enhancing experimental throughput.
Cell Culture Instructions
Cell Resuscitation
01. Preheat the complete culture medium in a 37°C water bath.
02. Thaw the cryovial in a 37°C water bath for 1–2 minutes.
03. Transfer the cryovials into a biosafety cabinet and wipe their surfaces with 70% ethanol.
04. Unscrew the cap of the cryovial and gently transfer the cell suspension into a sterile centrifuge tube containing 9 mL of complete culture medium.
05. Centrifuge at room temperature at 125g for 5–7 minutes, then discard the supernatant.
06. Resuspend the cell pellet in 5 mL of complete culture medium, and transfer the cell suspension to a T25 culture flask.
07. Transfer the cells to a 37°C incubator with 5% CO2 for culture.
08. Recommended passage ratio: 1/2 to 1/3; cells reach confluence in 2–3 days.
cell passage
01. When the cell confluence in the culture flask reaches 80%–90% or higher, cells can be passaged.
02. Remove the culture medium, PBS, and trypsin (0.25% Trypsin‑EDTA, Gibco 25200‑056) from the 4°C refrigerator, place them in a 37°C water bath, and once their temperature approaches 37°C, take them out. Spray the exterior of the bottles with 75% ethanol, then transfer them to a biosafety cabinet.
03. Remove the culture flask to be passaged from the incubator, spray the exterior of the flask with 75% ethanol, and place it inside a biosafety cabinet.
04. To avoid dispersing the cells, gently rinse the cells along the inner wall of the culture flask with PBS; discard the wash solution, and add 2 mL of PBS to the T25 flask.
05. Add the appropriate volume of trypsin (1.5 mL for a T75 flask, 0.5 mL for a T25 flask), and gently swirl the flask to ensure the enzyme evenly covers the cell‑covered bottom. Adjust the volume as needed based on the specific situation. After approximately 1–2 minutes, when most of the cells have detached, add the corresponding volume of complete culture medium to stop the digestion, then use a 5‑mL pipette to gently pipette up and down until all cells are fully detached.
06. Transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 300 × g for 5 minutes, and discard the supernatant.
07. Transfer 5 mL of complete culture medium to resuspend the cells, adjust the seeding ratio as needed, and replenish the culture flask with complete medium—add up to 13–15 mL for a T75 flask or 5 mL for a T25 flask—and supplement with 1% double antibiotic solution.
08. After capping the bottle and tightening the lid, gently shake the vial to ensure uniform cell suspension, then place it in a 37°C incubator with 5% CO2.
cell cryopreservation
01. Prepare the cryopreservation solution and pre-cool it in advance.
02. Ensure that the cells to be cryopreserved meet the cryopreservation requirements, and verify the following conditions under a microscope: healthy appearance and morphological characteristics, being in the late logarithmic growth phase, and showing no signs of contamination or senescence.
03. Digest and centrifuge the cells (refer to the subculture protocol for detailed steps).
04. Resuspend the cells in cryopreservation medium at 1 mL per tube, gently pipette up and down to ensure uniformity, and then aliquot into cryovials.
05. Place the cells in a programmed cooling box and freeze them in a −80°C freezer.
06. Subsequently, the cells are transferred to a liquid nitrogen tank for long-term storage.
Copyright © Elem Biotechnology (Wuhan) Co., Ltd.
LM