| Field | Specification |
|---|---|
| Organism | |
| Applications | |
| Tissue | |
| Disease | Derived from normal subcutaneous areolar and adipose tissue |
| Age | |
| Sex | |
| Growth properties | |
| Biosafety level | |
| Product format | Frozen |
| Storage | |
| Shipping | |
| Catalog no. (Mfr.) | |
| Main SKU |
Overview
This product is a stable CRISPR knockout cell line in which Aifm1 (Apoptosis Inducing Factor Mitochondria Associated 1) has been disrupted in the L929 mouse fibroblast background. Generated using CRISPR-Cas9 technology, this line provides an isogenic model for studying the role of AIFM1 in apoptosis, mitochondrial function, and redox biology. Cells are supplied frozen (1×10⁶ cells / 1.0 ml), classified as BSL-II, and grown as adherent, fibroblast-like monolayers.
CRISPR Knockout Design
- Target gene: Aifm1 (Apoptosis Inducing Factor Mitochondria Associated 1; mouse gene symbol)
- Knockout strategy: CRISPR-Cas9–mediated disruption of the Aifm1 locus with stable integration confirmed by puromycin selection
- Selection marker: Puromycin resistance; 7.0 µg/ml puromycin used for selection (add only after first passage post-thaw)
- Parental cell line: L929 — mouse fibroblast cell line derived from normal subcutaneous areolar and adipose connective tissue (Male, C3H/An, 100 days); adherent, fibroblast-like morphology
- Clone ID: T2-15-3 (negative clone designation)
Gene Background
AIFM1 (Apoptosis Inducing Factor Mitochondria Associated 1), also known as AIF, is a mitochondrial flavoprotein encoded by a nuclear gene and imported into the mitochondrial intermembrane space. Under normal physiological conditions, AIFM1 is involved in maintaining mitochondrial morphology and supporting oxidative phosphorylation through NADH oxidase activity. Upon receipt of apoptotic signals, AIFM1 is cleaved and released from the inner mitochondrial membrane, translocates to the nucleus, and triggers large-scale DNA fragmentation and chromatin condensation through a caspase-independent mechanism. Mutations and dysregulation of AIFM1 have been associated with mitochondrial encephalomyopathy and Cowchock syndrome, and the protein has been studied in the context of neurodegeneration, cancer cell death, and ischemia-reperfusion injury. The mouse Aifm1 gene is located on the X chromosome.
Cell Culture Specifications
- Recommended vessels
- PriCoat™ T25 Flasks (G299) or Applied Cell Extracellular Matrix (G422)
- Culture medium
- RPMI 1640 Medium (TM503) + 10% FBS (do not heat-inactivate) + 1% Penicillin/Streptomycin Solution (G255)
- Culture conditions
- 37.0°C, 5% CO₂
- Selection
- 7.0 µg/ml Puromycin (G264); add only after first passage post-thaw
- Growth properties
- Adherent, fibroblast-like
- Organism
- Mouse (M. musculus)
- Tissue origin
- Connective Tissue (subcutaneous areolar and adipose tissue)
- Biosafety level
- BSL-II
- Format
- Frozen; 1×10⁶ cells / 1.0 ml
Knockout is confirmed by the presence of frameshift-inducing INDELs, verified by Sanger Sequencing.
Research Applications
- Apoptosis pathway analysis: Investigate caspase-independent cell death mechanisms by comparing Aifm1 KO L929 cells to the isogenic parental line under apoptotic stimuli (e.g., H₂O₂, staurosporine, serum withdrawal).
- Mitochondrial function studies: Profile mitochondrial membrane potential (ΔΨm), ATP production, and oxygen consumption rate (OCR) in the absence of functional AIFM1 using standard respirometry assays.
- Drug sensitivity screening: Screen compounds targeting the intrinsic apoptotic pathway in the Aifm1 KO background; use the L929 parental line as the isogenic control.
- Redox biology: Quantify intracellular ROS levels and oxidative stress markers (e.g., lipid peroxidation, glutathione) in AIFM1-deficient cells.
- Rescue experiments: Re-express wild-type or mutant AIFM1 constructs in the KO background to investigate structure-function relationships.
Important Notes
- Selection drug timing: Add puromycin (7.0 µg/ml) to culture medium only after the first passage post-thaw to allow cells to recover from freeze-thaw conditions.
- Donor/source: Parental L929 cells are derived from normal subcutaneous areolar and adipose connective tissue of a male C3H/An mouse (approximately 100 days of age).
- Viability warranty: Cell viability upon thaw is warranted for 30 days following shipment when handled according to abm's cell handling guidelines.
- Intended use: For laboratory research use only. Not intended for diagnostic, therapeutic, or clinical applications.
- Thaw cells quickly in a 37°C water bath while agitating gently (maximum 2 minutes). The vial cap should be kept above the water level to minimize the risk of contamination.
- Decontaminate the vial by spraying and wiping the exterior of the vial with 70% ethanol. From this point onwards, all operations should be strictly carried out inside a biological safety cabinet using aseptic conditions.
- Transfer the cell suspension into a 15ml sterile conical tube containing 5ml of pre-warmed, complete growth media. Centrifuge cells at 125xg for 5-7 minutes.
- Aspirate the supernatant without disturbing the cell pellet. Re-suspend the cell pellet in the recommended pre-warmed, complete growth media and dispense into a T25 culture flask.
- Incubate the cells at the recommended conditions.
- Aspirate the culture media, and add 2-3ml of pre-warmed 0.25% Trypsin-EDTA to the culture vessel.
- Observe the cells under a microscope to confirm detachment (typically within 2-10 minutes). Cells that are difficult to detach can be put in 37°C, for several minutes to facilitate detachment.
- Neutralize Trypsin-EDTA by adding an equal volume of the complete growth media into the culture vessel.
- Transfer the culture suspension into a sterile centrifuge tube, and centrifuge at 125xg for 5 minutes. The actual centrifuge duration and speed may vary depending on the cell type.
- Aspirate the supernatant, and re-suspend the pellet with pre-warmed fresh complete growth media. Add appropriate aliquots of the cell suspension to new culture vessels, as desired.
- Incubate the cells at the recommended conditions.
How should I handle live cells once I receive them?
https://www.abmgood.com/immortalized-cells-documents.html
Following these guidelines will help ensure optimal cell viability and performance.
What is your warranty or return policy?
Please refer to the following link for full information:
https://www.abmgood.com/terms
For additional questions, our Order team is happy to assist and can be reached at order@abmgood.com.
How many times can cells divide?
Primary cells have a limited lifespan and will undergo a finite number of population doublings before entering senescence. The exact number varies by cell type and culture conditions.
Immortalized cell lines are capable of extended or indefinite proliferation under proper culture conditions, although growth characteristics may vary between lines.
Do I need Applied Cell Extracellular Matrix (G422) if I am using PriCoat™ flasks?
Cell line sourcing and selection (species, tissue, and disease model matching) · Stable cell line engineering (overexpression, knockdown, knockout via CRISPR/Cas9, shRNA, sgRNA) · Reporter gene integration (GFP, RFP, luciferase, fluorescent/bioluminescent constructs) · Genome editing and knockin (point mutations, tagged endogenous proteins, conditional alleles) · Inducible expression systems (Tet-On/Off and regulatable constructs) · Drug resistance marker selection (puromycin, G418, hygromycin, and others) · Custom growth and media optimisation for specific assay requirements · Scale-up production for high-throughput screening campaigns · Authentication and QC services (STR profiling, mycoplasma testing, viability assessment). Talk to a Scientist or contact support@biohippo.com.
