SDF 1a Mouse
Description
Introduction to SDF-1α Mouse
Stromal cell-derived factor-1 alpha (SDF-1α), also known as CXCL12, is a chemokine that plays a crucial role in various biological processes, including cell migration, proliferation, and survival. It is highly conserved across species, with only one amino acid substitution between the mature human and mouse proteins . This article will delve into the characteristics, functions, and research findings related to SDF-1α in mice.
Structure and Function
SDF-1α is encoded by the CXCL12 gene and arises from alternative splicing, resulting in a protein of 89 amino acid residues. It signals through the G protein-coupled receptor CXCR4 to recruit activated leukocytes . SDF-1α is known for its chemoattractant properties, affecting T-lymphocytes, monocytes, pro- and pre-B cells, but not neutrophils .
3.1. Immune System
SDF-1α is essential for B-lymphopoiesis and myelopoiesis. Mice lacking SDF-1 or CXCR4 exhibit impaired lymphoid organ development and function . In NZB/W mice, SDF-1α promotes the migration and proliferation of peritoneal B1a lymphocytes, which are involved in autoimmune responses .
3.2. Pancreatic β-Cell Survival
SDF-1α has been shown to promote pancreatic β-cell survival by activating Akt, a key signaling pathway for cell survival. This protective effect is significant in models of diabetes, where SDF-1α overexpression can mitigate streptozotocin-induced β-cell apoptosis .
3.3. Neuroprotection
In the context of neural injury, SDF-1α secreted by multipotent stromal cells can protect mouse neural progenitor cells through the CXCR7 receptor, suggesting a neuroprotective role .
3.4. Vascular and Neuronal Development
SDF-1α is crucial for vascular development and neuronal patterning in the central nervous system. Its absence leads to abnormalities in these processes .
4.1. Chemotactic Response in NZB/W Mice
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SDF-1α Concentration: As little as 50 ng/ml is effective in triggering the migration of peritoneal B1a lymphocytes from NZB/W mice, compared to 1 μg/ml needed for control mice .
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Actin Polymerization: SDF-1α induces higher levels of actin polymerization in NZB/W mice compared to controls, indicating enhanced sensitivity .
4.2. Protection Against Diabetes
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Akt Activation: Overexpression of SDF-1α in β-cells enhances Akt phosphorylation, promoting cell survival and resistance to streptozotocin-induced diabetes .
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Glucose Levels: RIP-SDF-1 mice maintain lower glucose levels compared to wild-type mice after streptozotocin injection .
4.3. Neuroprotection
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CXCR7 Signaling: SDF-1α protects mouse neural progenitor cells via CXCR7, not CXCR4, during hypoxia .
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Cell Survival: Knockdown of SDF-1α in multipotent stromal cells reduces the protective effect on neural progenitor cells .
Table 1: Biological Roles of SDF-1α
Table 2: Research Findings in NZB/W Mice
| Parameter | NZB/W Mice | Control Mice |
|---|---|---|
| SDF-1α Concentration for Migration | 50 ng/ml | 1 μg/ml |
| Actin Polymerization | Higher | Lower |
Table 3: Protection Against Diabetes
Product Specs
SDF-1, also known as stromal cell-derived factor-1, is a chemokine that plays a crucial role in directing cell migration and coordinating various cellular processes. It exists in two forms, SDF-1α (CXCL12a) and SDF-1β (CXCL12b), generated by alternative splicing of the CXCL12 gene. SDF-1 belongs to the CXC chemokine family, characterized by a specific arrangement of cysteine residues. It exhibits potent chemotactic activity, attracting lymphocytes and guiding the movement of hematopoietic cells during development. Notably, SDF-1 is essential for the homing of hematopoietic cells to the bone marrow during embryogenesis. Mice lacking the CXCL12 gene exhibit lethality either before or shortly after birth. Furthermore, SDF-1α influences neuronal electrophysiology. Its expression is observed in various tissues, including the brain, thymus, heart, lung, liver, kidney, spleen, and bone marrow. The primary receptor for SDF-1 is CXCR4, previously termed fusin. While this interaction was once considered exclusive, recent studies suggest that SDF-1 may also bind to the CXCR7 receptor. The CXCL12 gene is located on human chromosome 10. Both CXCL12 and CXCR4 exhibit high sequence identity between humans and mice, with 99% and 90% similarity, respectively.
Recombinant Mouse Stromal Cell-Derived Factor-1 alpha is produced in E. coli. It is a non-glycosylated polypeptide chain comprised of 68 amino acids, with a molecular weight of 8 kDa. This protein is purified using proprietary chromatographic methods.
This product appears as a sterile, white powder that has been lyophilized (freeze-dried).
The protein solution undergoes sterile filtration and is then lyophilized from an aqueous solution containing 0.1% Trifluoroacetic Acid (TFA).
To reconstitute the lyophilized Stromal Cell-Derived Factor-1a, it is recommended to dissolve it in sterile 18MΩ-cm H2O at a concentration of at least 100µg/ml. The reconstituted solution can be further diluted in other aqueous solutions.
Lyophilized SDF-1a remains stable at room temperature for up to 3 weeks. However, for long-term storage, it is recommended to store it in a desiccated state below -18°C. Once reconstituted, CXCL12 should be stored at 4°C for 2-7 days. For extended storage, adding a carrier protein like 0.1% HSA or BSA is advisable. Avoid repeated freeze-thaw cycles to maintain protein stability.
The purity of this product is determined by SDS-PAGE analysis and is greater than 95%.
The biological activity of this product is determined by its chemoattractant properties. It can effectively chemoattract human peripheral blood monocytes at concentrations of 50-100 ng/ml. This corresponds to a specific activity of 10,000-20,000 IU/mg.
Q&A
What is SDF-1α and how does it differ from SDF-1β in mouse models?
SDF-1α and SDF-1β are members of the chemokine alpha subfamily lacking the ELR domain, initially identified from mouse bone marrow stromal cell lines. Both variants are encoded by a single gene (CXCL12) and arise through alternative splicing. The mouse SDF-1α encodes a precursor protein of 89 amino acid residues, while SDF-1β encodes a 93 amino acid precursor. These proteins are identical except for four additional amino acid residues present in the carboxy-terminus of SDF-1β that are absent in SDF-1α .
Methodological approach to studying the variants: To distinguish between these variants in experimental work, researchers should employ isoform-specific RT-PCR primers targeting the unique regions of each splice variant, or use antibodies that can differentiate between the two forms for protein analysis.
How conserved is SDF-1α between mouse and human systems?
SDF-1α demonstrates remarkable evolutionary conservation between species. The mature SDF-1α proteins in mouse and human differ by only one amino acid substitution, representing approximately 99% sequence identity . This exceptional conservation suggests critical functional roles maintained throughout mammalian evolution.
What are the primary signaling pathways activated by mouse SDF-1α?
Mouse SDF-1α signals primarily through the G protein-coupled receptor CXCR4. This interaction triggers intracellular signaling cascades that regulate cell migration, proliferation, and survival . The signaling mechanisms include:
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Activation of G protein-coupled pathways
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Phosphorylation of multiple intracellular targets
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Mobilization of intracellular calcium
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Activation of PI3K/Akt and MAP kinase pathways
Experimental approach: When investigating SDF-1α signaling, researchers should employ phospho-specific antibodies to track activation of downstream effectors, calcium flux assays to measure immediate responses, and selective pathway inhibitors to delineate the contribution of specific signaling branches to observed biological effects.
What are the optimal methods for quantifying SDF-1α protein levels in mouse samples?
Several complementary approaches can be used to quantify SDF-1α in mouse samples:
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ELISA: Mouse SDF-1α solid-phase sandwich ELISA kits can measure SDF-1α in serum, plasma, or cell culture medium with high sensitivity . This method allows precise quantification but provides no spatial information.
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Western blotting: SDF-1α can be detected using SDS-PAGE under reducing conditions. The protein typically appears as a band at approximately 7-8 kDa .
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Immunohistochemistry: For tissue localization, immunostaining provides crucial spatial information about SDF-1α distribution.
Methodological considerations: When analyzing SDF-1α in blood, researchers must account for its presence both as a soluble protein and within platelets. Sample preparation methods significantly affect measurements - approximately 2 ng of soluble SDF-1α can be detected in ~1 mL of blood from wild-type mice .
| Sample Type | Typical SDF-1α Concentration | Detection Method | Special Considerations |
|---|---|---|---|
| Mouse plasma | 2-4 ng/mL (wild-type) | ELISA | Avoid platelet activation during collection |
| Bone marrow fluid | 100-400 pg/mL | ELISA | Rapid collection to prevent degradation |
| Cell culture supernatant | Variable | ELISA/Western blot | Standardize collection timing |
How can researchers effectively evaluate SDF-1α-induced chemotaxis in mouse cells?
To quantitatively assess SDF-1α-induced chemotaxis:
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Transwell migration assays: Place target cells (e.g., lymphocytes or hematopoietic progenitors) in the upper chamber with recombinant mouse SDF-1α in the lower chamber at various concentrations. After incubation, quantify migrated cells by flow cytometry or microscopy .
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Real-time cell tracking: For detailed migration dynamics, time-lapse microscopy with automated cell tracking software provides information on directionality, velocity, and persistence.
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In vivo migration assays: Labeled cells can be injected into mice and their SDF-1α-dependent homing to specific tissues tracked using intravital microscopy or post-mortem analysis.
Technical parameters: Recombinant mouse CXCL12/SDF-1α chemoattracts BaF3 mouse pro-B cells transfected with human CXCR4 with an ED50 of 0.15-0.6 ng/mL . This information is crucial for designing dose-response experiments with appropriate concentration ranges.
What approaches can resolve contradictory data regarding SDF-1α expression in different mouse tissues?
Contradictory data regarding SDF-1α expression can be resolved through:
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Multi-level analysis: Examine both mRNA (using RT-qPCR) and protein (using ELISA and immunohistochemistry) to identify post-transcriptional regulation events .
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Strain-specific differences: Acknowledge and control for strain backgrounds when comparing data across studies. SDF-1α responses vary significantly between strains (e.g., NZB versus other backgrounds) .
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Age and disease state considerations: SDF-1α expression patterns change with age and disease progression. In Gata1low mice (a myelofibrosis model), SDF-1α mRNA in femurs decreases with age while protein deposition increases .
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Cell-specific expression analysis: Single-cell RNA sequencing or cell sorting prior to analysis can resolve seemingly contradictory tissue-level data by identifying cell-specific expression patterns.
How does SDF-1α functionally contribute to mouse development?
SDF-1α plays critical roles in multiple developmental processes, as evidenced by severe defects in knockout models:
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Hematopoietic development: SDF-1α is essential for B-lymphopoiesis and myelopoiesis .
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Cardiovascular development: SDF-1α knockout mice exhibit impaired vascular development and cardiogenesis .
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Neuronal development: SDF-1α regulates neuronal cell migration and patterning in the central nervous system .
Methodological approach for developmental studies: Conditional knockout systems are preferable to global knockouts (which are often embryonic lethal) when studying specific developmental processes. These allow temporal and tissue-specific deletion of SDF-1α to assess stage-specific requirements.
What are the altered SDF-1α/CXCR4 dynamics in mouse models of myelofibrosis?
In Gata1low mice, which model myelofibrosis, significant alterations in the SDF-1α/CXCR4 axis include:
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Elevated plasma SDF-1α protein levels (5-times higher than normal in younger animals)
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Progressive increase in bone marrow SDF-1α protein deposition with age, despite decreased mRNA expression
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Lower expression of CXCR4 mRNA and protein on stem/progenitor cells
Translational significance: These abnormalities mirror those observed in human primary myelofibrosis patients, where similar alterations in the SDF-1α/CXCR4 axis contribute to increased stem/progenitor cell trafficking . This finding validates the Gata1low model for studying therapeutic interventions targeting this pathway.
How does SDF-1α contribute to autoimmune pathology in mouse models?
In New Zealand Black/New Zealand White (NZB/W) mice that develop lupus-like symptoms:
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Peritoneal B1a lymphocytes show abnormally high sensitivity to SDF-1α, attributed to the NZB genetic background
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SDF-1α is constitutively produced in the peritoneal cavity and spleen, and in advanced disease, by podocytes in glomeruli during nephritis
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Blocking SDF-1α with antagonists or antibodies:
Therapeutic implications: These findings suggest SDF-1α as a potential therapeutic target in autoimmune conditions involving dysregulated B cell activity and tissue-specific inflammation.
How can manipulation of the SDF-1α/CXCR4 axis be optimized for bone marrow transplantation in mice?
Optimizing SDF-1α/CXCR4 manipulation for bone marrow transplantation involves several strategic approaches:
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Pre-conditioning recipient mice: Increased SDF-1α production following DNA damage improves bone marrow recovery and facilitates stem cell engraftment . Radiation or chemotherapy timing can be adjusted to maximize this effect.
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Donor cell CXCR4 modulation: Transient upregulation of CXCR4 on donor cells can enhance homing efficiency to SDF-1α gradients in recipient bone marrow.
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Post-transplantation manipulation: Administering agents that stabilize SDF-1α or enhance its signaling during the early post-transplant period may improve engraftment outcomes.
Experimental evidence: Inactivation of CXCR4 function with neutralizing antibodies impairs stem/progenitor cell engraftment in bone marrow , highlighting the essential role of this pathway in transplantation success.
What methodological approaches can distinguish the unique functions of SDF-1α versus SDF-1β in mouse models?
To distinguish the specific functions of these highly similar splice variants:
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Isoform-specific genetic models:
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CRISPR/Cas9-mediated introduction of stop codons to selectively eliminate either isoform
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Transgenic mice expressing only SDF-1α or SDF-1β under native regulatory elements
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Differential expression analysis:
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Single-cell RNA-seq to identify cells/tissues with predominant expression of one isoform
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Temporal analysis during development or disease progression to identify isoform-specific expression patterns
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Structure-function studies:
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Administration of recombinant proteins with mutations in the differential C-terminal region
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Generation of isoform-specific blocking antibodies targeting the unique C-terminal region of SDF-1β
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Research design considerations: When conducting structure-function studies, researchers should consider that the four additional amino acids in SDF-1β may affect not only receptor binding but also interactions with extracellular matrix components and proteolytic stability.
How do genetic background differences affect SDF-1α biology in diverse mouse strains?
Mouse strain differences significantly impact SDF-1α biology:
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Strain-specific sensitivity: New Zealand Black (NZB) mice show intrinsically higher SDF-1α responsiveness in peritoneal B1a lymphocytes compared to other strains .
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Cell type specificity: Strain differences may be cell-type specific - the hypersensitivity seen in NZB mice is not observed in other B lymphocyte subpopulations .
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Cytokine interactions: The modulatory effect of IL-10 on SDF-1α responses varies between strains .
Methodological implications for experimental design:
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Always report complete strain information in publications
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Use appropriate genetic background controls when studying transgenic or knockout models
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Consider backcrossing strategy when transferring mutations between strains
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Validate key findings across multiple genetic backgrounds when possible
What are the latest approaches for manipulating SDF-1α in temporal and spatial dimensions for mouse tissue engineering applications?
Advanced approaches for precise SDF-1α manipulation include:
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Biomaterial-based delivery systems:
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Genetic engineering approaches:
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Cell-based delivery using engineered cells with inducible SDF-1α expression
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AAV-mediated localized gene delivery with tissue-specific promoters
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Optogenetic or chemically-inducible expression systems for precise temporal control
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Targeting natural regulatory mechanisms:
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Inhibitors of proteases that degrade SDF-1α to extend signaling duration
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Modulators of SDF-1α transcription to enhance endogenous production
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Technical example: Nanoparticle-modified chitosan-agarose-gelatin scaffolds have been developed for sustained release of SDF-1 and BMP-2, demonstrating the feasibility of complex delivery systems for tissue engineering applications .
What is the optimal workflow for analyzing SDF-1α expression at both mRNA and protein levels in mouse tissues?
A comprehensive workflow integrating multiple techniques:
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Sample collection and processing:
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Flash-freeze tissues in liquid nitrogen immediately after collection
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For bone marrow, either flush bones with ice-cold PBS or process entire femurs
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For blood, use platelet-poor plasma to avoid platelet-derived SDF-1α contamination
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mRNA analysis:
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Extract RNA using methods optimized for each tissue type
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Perform RT-qPCR with validated primer sets for SDF-1α and appropriate housekeeping genes
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Consider analyzing both total SDF-1 and specific splice variants
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Protein analysis:
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Integrated data analysis:
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Correlate mRNA and protein levels to identify post-transcriptional regulation
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Integrate with functional assays (e.g., chemotaxis) to determine biological significance
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Validation criteria: Research has established that in wild-type mice, liver has the highest SDF-1α mRNA expression (10-fold higher than bone marrow and 100-fold higher than spleen) , providing a reference point for validation.
How can researchers distinguish between different pools of SDF-1α protein in mouse samples?
SDF-1α exists in multiple physiological pools that require different analytical approaches:
Methodological approach: To comprehensively profile SDF-1α distribution, researchers should employ differential extraction techniques combined with pool-specific quantification methods. For example, comparing SDF-1α levels in platelet-rich versus platelet-poor plasma reveals the platelet-sequestered fraction.
Product Science Overview
SDF-1α was first identified as the pre-B-cell growth-stimulating factor (PBSF) in the mouse bone marrow-derived stromal cell line, PA6 . It is one of two splice variants produced by cells when stimulated by inflammatory cytokines such as TNF, IL-1, or LPS . The human and mouse versions of SDF-1α share 99% sequence identity . Recombinant mouse SDF-1α is a non-glycosylated protein containing 68 amino acids and has a molecular mass of 7.9 kDa .
SDF-1α signals through the G protein-coupled receptor, CXCR4, to recruit activated leukocytes . This signaling pathway is essential for various biological processes, including immune response, tissue repair, and development. SDF-1α is up-regulated in diseases characterized by tissue hypoxia, such as myocardial infarction and ischemic cardiomyopathy .
Due to its significant role in disease mechanisms, SDF-1α is a potential biomarker for conditions characterized by tissue hypoxia . Researchers have developed novel recombinant antibodies specific to full-length SDF-1α to facilitate its investigation in biomarker studies . These antibodies have been used to quantify full-length SDF-1α in blood, providing insights into its kinetics and role in various conditions .
Recombinant mouse SDF-1α is typically lyophilized from a concentrated sterile solution containing 10 mM acetic acid . The lyophilized product is stable at -20°C, and reconstituted material should be aliquoted and frozen at -20°C for long-term storage . It is recommended to add a carrier protein, such as 0.1% HSA or BSA, for extended storage .
