U2OS-CRISPR-SNAPf-Nup133 Cells

USUSD$800.00*

Products are shipped frozen on dry ice in cryotubes. Each cryotube typically contains 3 × 10⁶ cells for adherent lines or 5 × 10⁶ cells for suspension lines (refer to the batch CoA for details).

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cryovial

產品編號： 300666

安全資料表

產品說明書

分析證明書 (CoA)

說明	U2OS-CRISPR-SNAPf-Nup133 is a genetically engineered human osteosarcoma cell line derived from the parental U2OS background in which the endogenous NUP133 locus has been modified using CRISPR/Cas9-mediated genome editing to encode a C-terminal SNAPf tag. NUP133 is a core component of the Y-complex (NUP107-160 complex), a structural subcomplex essential for nuclear pore complex (NPC) assembly and maintenance. By introducing the SNAPf coding sequence in-frame at the endogenous locus, the fusion protein is expressed under native regulatory control, preserving physiological expression levels and subcellular localization. The SNAPf tag is a fast-labeling variant of the SNAP-tag, an engineered O6-alkylguanine-DNA alkyltransferase that covalently reacts with benzylguanine-conjugated substrates. This enables highly specific and versatile fluorescent labeling of Nup133 in live or fixed cells using cell-permeable or impermeable SNAP substrates. In U2OS-CRISPR-SNAPf-Nup133 cells, the fusion protein localizes to the nuclear envelope in a punctate pattern characteristic of nuclear pore complexes. Because tagging occurs at the endogenous locus, NPC stoichiometry and architecture are minimally perturbed, making this model suitable for quantitative super-resolution microscopy, single-molecule tracking, and kinetic analyses of NPC assembly and turnover. This cell line provides a robust platform for studying nuclear transport, nucleocytoplasmic trafficking dynamics, NPC biogenesis during interphase and post-mitotic nuclear reassembly, and structural organization of the Y-complex within the pore scaffold. The U2OS background offers flat morphology and large nuclei, facilitating high-resolution imaging. U2OS-CRISPR-SNAPf-Nup133 cells are particularly well suited for pulse-chase labeling experiments, correlative light and electron microscopy, and multicolor imaging approaches in combination with additional endogenously tagged nucleoporins or transport factors.
生物體	Human
組織	Bone
疾病	Osteosarcoma

年齡	15 years
性別	Female
族裔	Caucasian
形態學	Epithelial-like
生長特性	Adherent

引用	U2OS-CRISPR-SNAPf-Nup133 (Cytion catalog number 300666)
生物安全等級	1
NCBI_TaxID	9606
存款人	The Ellenberg Lab (EMBL)
轉基因狀態	GMO-S1: This human osteosarcoma cell line (U2OS-CRISPR-SNAPf-Nup133) contains a CRISPR-introduced SNAPf-Nup133 fusion, enabling fluorescent tagging of the Nup133 nucleoporin. The insert is stably present. This classification applies only within Germany and may differ elsewhere.

蛋白質表達	Nup133, SNAPf-tag

培養基	McCoys 5a, w: 3.0 g/L Glucose, w: stable Glutamine, w: 2.0 mM Sodium pyruvate, w: 2.2 g/L NaHCO3 (Cytion article number 820200a)
營養補充品	Supplement the medium with 10% FBS, 3.0 g/L Glucose, stable Glutamine, 2.0 mM Sodium pyruvate, 2.2 g/L NaHCO3, 1% NEAA
解離試劑	Accutase
傳代培養	Remove the old medium from the adherent cells and wash them with PBS that lacks calcium and magnesium. For T25 flasks, use 3-5 ml of PBS, and for T75 flasks, use 5-10 ml. Then, cover the cells completely with Accutase, using 1-2 ml for T25 flasks and 2.5 ml for T75 flasks. Let the cells incubate at room temperature for 8-10 minutes to detach them. After incubation, gently mix the cells with 10 ml of medium to resuspend them, then centrifuge at 300xg for 3 minutes. Discard the supernatant, resuspend the cells in fresh medium, and transfer them into new flasks that already contain fresh medium.
冷凍培養基	As a cryopreservation medium, we use complete growth medium (including FBS) + 10% DMSO for adequate post-thaw viability, or CM-1 (Cytion catalog number 800100), which includes optimized osmoprotectants and metabolic stabilizers to enhance recovery and reduce cryo-induced stress.
細胞解凍與培養	Confirm that the vial remains deeply frozen upon delivery, as cells are shipped on dry ice to maintain optimal temperatures during transit. 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. 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. Perform all subsequent steps under sterile conditions in a flow hood, disinfecting the cryovial with 70% ethanol before opening. 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. Centrifuge the mixture at 300 x g for 3 minutes to separate the cells and carefully discard the supernatant containing residual freezing medium. 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. Adhere to established subculture protocols for continued growth and maintenance of the cell line, ensuring reliable experimental outcomes.
培養環境	37°C, 5% CO₂, humidified atmosphere.
運送條款	Cryopreserved cell lines are shipped on dry ice in validated, insulated packaging with sufficient refrigerant to maintain approximately −78 °C throughout transit. On receipt, inspect the container immediately and transfer vials without delay to appropriate storage.
儲存條件	For long-term preservation, place vials in vapor-phase liquid nitrogen at about −150 to −196 °C. Storage at −80 °C is acceptable only as a short interim step before transfer to liquid nitrogen.

不孕症	Mycoplasma contamination is excluded using both PCR-based assays and luminescence-based mycoplasma detection methods. To ensure there is no bacterial, fungal, or yeast contamination, cell cultures are subjected to daily visual inspections.