| Field | Specification |
|---|---|
| Organism | |
| Applications | |
| Tissue | |
| Disease | Abelson murine leukemia virus-induced tumor |
| 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 Araf (A-RAF proto-oncogene, serine/threonine kinase) has been disrupted in the RAW264.7 mouse macrophage background. The knockout enables loss-of-function investigation of ARAF's role in MAPK/ERK signaling and innate immune responses within a macrophage model. RAW264.7 cells are a widely used murine macrophage-like cell line derived from an Abelson murine leukemia virus–induced tumor (Balb/c, adult male). Cells grow as loosely adherent monolayers and are supplied frozen (BSL-II) with puromycin selection confirming stable knockout.
CRISPR Knockout Design
- Target gene: Araf (A-RAF proto-oncogene, serine/threonine kinase; mouse gene symbol)
- Knockout strategy: CRISPR-Cas9–mediated disruption of the Araf locus; stable integration confirmed by puromycin selection
- Selection marker: Puromycin resistance (add only after first passage post-thaw)
- Parental cell line: RAW264.7 — mouse macrophage cell line; adherent, loosely adherent; derived from ascites of an adult male Balb/c mouse bearing an Abelson murine leukemia virus–induced tumor
- Clone ID: T1-19
Gene Background
ARAF (A-RAF proto-oncogene) is a member of the RAF family of serine/threonine kinases, which also includes BRAF and CRAF (RAF1). RAF kinases are key effectors in the RAS–RAF–MEK–ERK mitogen-activated protein kinase (MAPK) cascade, linking extracellular growth factor signals to transcriptional regulation of cell proliferation, differentiation, and survival. While BRAF and CRAF have been more extensively characterized in cancer biology, ARAF is expressed in several tissues including macrophages and is involved in modulating MEK–ERK activation. In macrophages, MAPK signaling pathways including the ERK pathway are known to influence responses to inflammatory stimuli such as LPS. ARAF activity may contribute to the balance of inflammatory versus anti-inflammatory cytokine production.
Cell Culture Specifications
- Growth properties
- Adherent, loosely adherent
- Organism
- Mouse (M. musculus)
- Tissue origin
- Ascites (macrophage; Abelson murine leukemia virus–induced tumor)
- 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
- MAPK/ERK pathway analysis: Investigate the specific contribution of ARAF to MEK–ERK signaling in a macrophage context; compare phospho-ERK levels between Araf KO and parental RAW264.7 cells under basal and stimulated conditions.
- Innate immune signaling: Profile inflammatory cytokine production (e.g., TNFα, IL-6, IL-1β) in Araf-deficient vs. parental macrophages following LPS or other TLR stimulation.
- RAF isoform biology: Distinguish ARAF-dependent signaling from BRAF/CRAF-mediated ERK activation using this KO line alongside isoform-specific inhibitors.
- Drug screening: Use the Araf KO line as a negative control when screening pan-RAF or ARAF-selective inhibitors to confirm on-target activity.
- Macrophage polarization: Assess M1 (pro-inflammatory) vs. M2 (anti-inflammatory) polarization capacity in ARAF-deficient RAW264.7 cells under appropriate stimulation conditions.
Important Notes
- Loosely adherent growth: RAW264.7 cells are loosely adherent and may detach during routine media changes; use gentle aspiration and avoid disturbing the cell monolayer.
- Selection drug timing: Add puromycin to culture medium only after the first passage post-thaw to allow cells to recover from freeze-thaw conditions.
- Donor/source: RAW264.7 parental cells originate from ascites of an adult male Balb/c mouse bearing an Abelson murine leukemia virus–induced tumor.
- 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.
