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HK Mad2-LAP/H2B-mCherry
Tissue
子宮頸
Disease
癌
Synonyms
HeLa Kyoto Mad2-LAP and H2B-mCherry, HeLa Kyoto Mad2-LAP

Documents

HK Mad2-LAP/H2B-mCherry

Cell line · Product number 300920

The HK Mad2-LAP/H2B-mCherry cell line is a genetically engineered cell model extensively utilized for studying chromosome segregation and the spindle asse...

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01General information

Description

HK Mad2-LAP/H2B-mCherry 細胞系是一種經基因工程改造的細胞模型,廣泛用於研究有絲分裂期間的染色體分離及紡錘體組裝檢查點。這些細胞源自 HeLa Kyoto 細胞,這是一種最初從子宮頸癌中分離出的、具有高度穩定性的人類細胞系。 該細胞系的 HK Mad2-LAP(帶有 LAP 標記的 Mad2)特性,有助於對 Mad2 蛋白進行可視化觀察與功能分析;Mad2 蛋白是紡錘體組裝檢查點的關鍵組成部分,其作用是在所有染色體於中期板正確對齊之前,阻止後期開始。

透過整合 H2B-mCherry(即將組蛋白 H2B 標記上 mCherry 螢光蛋白),可實現細胞分裂過程中染色質動態的即時成像。 此特性使 HK Mad2-LAP/H2B-mCherry 細胞系成為高解析度活細胞成像技術的絕佳工具,可用於觀察人類細胞在各種實驗條件下之染色體運動與有絲分裂進程。 螢光標記的應用有助於精確追蹤與定量分析,從而為細胞週期調控與染色體穩定性的分子機制提供寶貴見解。

Tissue
子宮頸
Disease
癌
Synonyms
  • HeLa Kyoto Mad2-LAP and H2B-mCherry
  • HeLa Kyoto Mad2-LAP

02Classification

Organism
Human
Sex
Female
Growth properties
Adherent
GMO status
GMO-S1
GMO status details
GMO-S1: This HeLa Kyoto line contains Mad2-LAP and H2B-mCherry constructs enabling visualization of spindle checkpoint dynamics. This classification applies only within Germany and may differ elsewhere.
Biosafety level
1

03Characteristics

Morphology
呈馬賽克狀石塊形狀的類上皮細胞

04Identifiers

Citation
HK Mad2-LAP/H2B-mCherry(Cytion 型號 300920)
NCBI TaxID
9606
Cellosaurus
CVCL_1D65
Depositor
艾倫伯格實驗室(EMBL)

05Expression and susceptibility

Protein expression
Mad2-LAP/H2B-mCherry

06Handling and culture

Culture medium
DMEM,含 4.5 g/L 葡萄糖、4 mM L-谷氨醯胺、3.7 g/L NaHCO₃ 及 1.0 mM 丙酮酸鈉(Cytion 產品編號 820300a)
Incubation atmosphere

37°C,5% CO₂,加濕環境。

Supplements
在培養基中加入 10% 的胎牛血清(FBS)
Dissociation reagent
Accutase
Subculturing
從貼壁細胞中移除舊培養基,並用不含鈣和鎂的PBS洗滌細胞。T25培養瓶使用3-5毫升PBS,T75培養瓶則使用5-10毫升。 接著,用 Accutase 完全覆蓋細胞,T25 培養瓶使用 1–2 毫升,T75 培養瓶則使用 2.5 毫升。將細胞於室溫下孵育 8–10 分鐘,使其脫附。 孵育後,將細胞與 10 毫升培養基輕柔混合以使其重新懸浮,然後以 300xg 離心 3 分鐘。棄去上清液,將細胞重新懸浮於新鮮培養基中,並轉移至已預先加入新鮮培養基的新培養瓶中。
Seeding density
1 × 10⁴ 個細胞/cm²
Fluid renewal
每週 2 至 3 次
Post-thaw recovery
解凍後,將細胞以 5 × 10⁴ 個細胞/cm² 的密度接種於培養皿中,並讓細胞從冷凍過程中恢復,並附著至少 24 小時。
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

透過基於 PCR 的檢測及基於發光原理的支原體檢測方法,排除支原體污染的可能性。

為確保無細菌、真菌或酵母菌污染,每日均會對細胞培養物進行目視檢查。

Freeze medium
作為冷凍保存培養基,我們使用完全生長培養基(含 FBS)+ 10% DMSO 以確保解凍後的存活率,或使用 CM-1 (Cytion 型號 800100),該培養基含有經優化的滲透保護劑和代謝穩定劑,可提升細胞恢復率並減輕冷凍誘導的壓力。
Storage conditions

若需長期保存,請將試管置於溫度約為 −150 至 −196 °C 的氣相液氮中。僅在轉移至液氮之前,可將試管暫時存放於 −80 °C 環境中,作為短期過渡步驟。

Shipping conditions

冷凍保存的細胞系會以乾冰冷藏,並裝於經驗證的隔熱包裝中,包裝內含有足夠的冷媒,以確保在運輸過程中溫度維持在約 −78 °C。收到貨物後,請立即檢查容器,並盡快將試管轉移至適當的儲存場所。

08Donor

Donor age
30年
Ethnicity
非裔美國人