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HK EGFP-LaminB1/H2B-mCherry
Tissue
子宫颈
Disease
癌症
Metastatic site
原发肿瘤部位(宫颈)
Synonyms
HeLa Kyoto EGFP-LaminB1 and H2B-mCherry

Documents

HK EGFP-LaminB1/H2B-mCherry

Cell line · Product number 300919

The HK EGFP-LaminB1/H2B-mCherry cell line is an Hela Kyoto derived in vitro model designed for real-time visualization of chromatin dynamics and nuclear a...

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01General information

Description

HK EGFP-LaminB1/H2B-mCherry 细胞系是一种源自京都大学的 Hela 体外模型,用于实时观察活细胞中染色质的动态和核结构。该细胞系表达两种荧光蛋白融合体:EGFP(增强型绿色荧光蛋白)与 Lamin B1 融合,mCherry(红色荧光蛋白)与组蛋白 H2B 融合。EGFP 与 Lamin B1 融合后,可以观察到核膜和核薄层,这些结构对维持细胞核的完整性和功能性至关重要。层蛋白是 V 型中间丝蛋白,在核内膜下形成网状结构,在核稳定性、染色质组织和基因调控中发挥关键作用。

另一方面,mCherry 标记的组蛋白 H2B 使细胞核内染色质的可视化成为可能。组蛋白是核小体的基本成分,参与将 DNA 组织成染色质,对 DNA 复制、修复和转录至关重要。H2B 上的 mCherry 标记能发出鲜艳的红色荧光,与 EGFP 的绿色荧光形成鲜明对比,可在活细胞实验中同时对核结构和染色质进行双重成像。这种细胞系常用于研究核机制、有丝分裂和基因组稳定性,可动态观察难以实时观察到的细胞过程。

Tissue
子宫颈
Disease
癌症
Metastatic site
原发肿瘤部位(宫颈)
Applications
核层板与染色质组织;Lamin B1动力学;H2B染色质成像;双色活细胞荧光;核力学;有丝分裂;基因组稳定性;核膜生物学
Synonyms
  • HeLa Kyoto EGFP-LaminB1 and H2B-mCherry

02Classification

Organism
Human
Sex
Female
Growth properties
Adherent
GMO status
GMO-S1
GMO status details
GMO-S1: This HeLa Kyoto line contains EGFP-Lamin B1 and H2B-mCherry constructs for imaging nuclear envelope and chromatin organization. This classification applies only within Germany and may differ elsewhere.
Biosafety level
1

03Characteristics

Morphology
具有镶嵌石形状的上皮样细胞
Cell type
上皮细胞

04Identifiers

Citation
HK EGFP-LaminB1/H2B-mCherry(Cytion 目录号 300919)
NCBI TaxID
9606
Cellosaurus
CVCL_UR41
Depositor
艾伦伯格实验室(EMBL)

05Expression and susceptibility

Protein expression
EGFP-LaminB1/H2B-mCherry
Products
组蛋白 H2B

06Handling and culture

Culture medium
DMEM,w:4.5 克/升葡萄糖,w:4 毫摩尔 L-谷氨酰胺,w:3.7 克/升 NaHCO3,w:1.0 毫摩尔丙酮酸钠(Cytion 编号 820300a)。
Incubation atmosphere

37°C, 5%CO2, 加湿环境。

Supplements
在培养基中添加 10% FBS
Dissociation reagent
Accutase
Subculturing
去除粘附细胞上的旧培养基,用不含钙和镁的 PBS 冲洗。T25 烧瓶用 3-5 毫升 PBS,T75 烧瓶用 5-10 毫升。然后用 Accutase 完全覆盖细胞,T25 烧瓶用 1-2 毫升,T75 烧瓶用 2.5 毫升。让细胞在室温下孵育 8-10 分钟,使其分离。孵育后,用 10 毫升培养基轻轻混合细胞使其重悬,然后用 300xg 离心 3 分钟。弃去上清液,用新鲜培养基重悬细胞,并将其转移到已装有新鲜培养基的新烧瓶中。
Seeding density
1 × 10⁴ 个细胞/平方厘米
Fluid renewal
每周 2 至 3 次
Post-thaw recovery
解冻后,将细胞以5×10⁴个细胞/平方厘米的密度接种于培养皿中,使细胞从冷冻过程中恢复并至少贴壁24小时。
Thawing and culturing
  1. Confirm that the vial remains deeply frozen upon delivery, as cells are shipped on dry ice to maintain optimal temperatures during transit.

  2. Upon receipt, either store the cryovial immediately at temperatures below -150°C to ensure the preservation of cellular integrity, or proceed to step 3 if immediate culturing is required.

  3. For immediate culturing, swiftly thaw the vial by immersing it in a 37°C water bath with clean water and an antimicrobial agent, agitating gently for 40-60 seconds until a small ice clump remains.

  4. Perform all subsequent steps under sterile conditions in a flow hood, disinfecting the cryovial with 70% ethanol before opening.

  5. Carefully open the disinfected vial and transfer the cell suspension into a 15 ml centrifuge tube containing 8 ml of room-temperature culture medium, mixing gently.

  6. Centrifuge the mixture at 300 x g for 3 minutes to separate the cells and carefully discard the supernatant containing residual freezing medium.

  7. Gently resuspend the cell pellet in 10 ml of fresh culture medium. For adherent cells, divide the suspension between two T25 culture flasks; for suspension cultures, transfer all the medium into one T25 flask to promote effective cell interaction and growth.

  8. Adhere to established subculture protocols for continued growth and maintenance of the cell line, ensuring reliable experimental outcomes.

07Quality control and cryopreservation

Sterility

使用基于 PCR 的检测方法和基于发光的支原体检测方法排除支原体污染。

为确保没有细菌、真菌或酵母菌污染,每天都要对细胞培养物进行目视检查。

Freeze medium
我们使用完全生长培养基(包括 FBS)+10% DMSO 或 CM-1(Cytion 商品目录编号 800100)作为冷冻保存培养基,以获得足够的解冻后存活率,CM-1(Cytion 商品目录编号 800100)含有优化的渗透保护剂和代谢稳定剂,可提高恢复能力并减少冷冻引起的应激。
Storage conditions

如需长期保存,可将样品瓶放入气相液氮中,温度约为 -150 至 -196 ℃。在-80 °C下保存只能作为转入液氮前的短暂过渡。

Shipping conditions

冷冻保存的细胞系用干冰装运,并用经过验证的隔热包装和足够的制冷剂在整个运输过程中保持约 -78 °C 的温度。收到货后,应立即检查容器,并立即将小瓶转移到适当的储藏室。

08Donor

Donor age
30年
Ethnicity
非洲裔美国人