CD9 Antibody, FITC conjugated

What is CD9 and what cellular functions does it regulate?

CD9, also known as motility-related protein-1 (MRP-1), p24, or Tspan-29, is a member of the tetraspanin superfamily. It is an integral membrane protein that functions through associations with integrins and other membrane proteins. CD9 regulates multiple cellular processes including sperm-egg fusion, platelet activation and aggregation, and cell adhesion . On the molecular level, CD9 forms both homotypic associations and heterotypic associations with other tetraspanins, certain integrins, and MHC class II proteins . CD9 is present at the cell surface of oocytes and plays a key role in sperm-egg fusion, possibly by organizing multiprotein complexes and creating the appropriate membrane morphology required for fusion . In myoblasts, CD9 associates with CD81 and PTGFRN to inhibit myotube fusion during muscle regeneration . Additionally, in macrophages, CD9 associates with integrins and prevents their fusion into multinucleated giant cells that specialize in ingesting complement-opsonized large particles .

What cell types express CD9 and can be detected with CD9-FITC antibodies?

CD9 is expressed on a remarkably diverse range of cell types, making CD9-FITC antibodies valuable tools for numerous research applications. The protein is found on both hematopoietic and non-hematopoietic cells, including stromal cells, megakaryocytes, platelets, B and T lymphocytes, dendritic cells, endothelial cells, mast cells, eosinophils, and basophils . This broad expression pattern explains why CD9 has been implicated in so many biological processes. Flow cytometry experiments have successfully detected CD9 on human peripheral blood platelets using FITC-conjugated anti-CD9 antibodies . The detection of CD9 on platelets is particularly significant as CD9 modulates platelet aggregation when activated by various agonists . When selecting cell types for your experiments, it is important to consider that expression levels may vary substantially between different tissues and cellular activation states.

What are the key technical specifications of CD9-FITC antibodies?

CD9-FITC antibodies are available from several manufacturers with different technical specifications. These antibodies typically have FITC (Fluorescein Isothiocyanate) as the conjugated fluorochrome with excitation/emission maxima wavelengths of approximately 498 nm and 526 nm . They are generally supplied in liquid form in PBS buffer with 0.1% sodium azide . The recommended storage condition is 2-8°C with protection from light exposure, and most products remain stable for one year after shipment when properly stored . For flow cytometry applications, the suggested dilution is typically 10 μl per 10^6 cells in a 100 μl suspension or 10 μl per 100 μl of whole blood, though optimal dilutions should be determined by each laboratory for specific applications . It is important to note that different clones (such as MEM-61, EM-04, 209306, ALB6, and HI9a) may have different performance characteristics depending on your experimental system.

How should CD9-FITC antibodies be validated before experimental use?

Validation of CD9-FITC antibodies is crucial for ensuring reliable experimental results. A comprehensive validation approach should include positive and negative controls. For positive controls, use cell types known to express CD9, such as platelets, which have been consistently shown to express high levels of CD9 . Peripheral blood platelets serve as excellent positive controls as demonstrated in flow cytometry validation experiments by multiple manufacturers . For negative controls, use appropriate isotype control antibodies that match the host species and isotype of your CD9-FITC antibody, such as Mouse IgG1 or IgG2b depending on the specific clone . When analyzing human samples, the ALB6 clone has been validated for CD9 detection on platelets and shown to modulate platelet aggregation . For mouse samples, the EM-04 clone has been cited in published research for flow cytometry applications . Cross-reactivity testing should be performed if working with species other than those explicitly tested by the manufacturer, with special attention to the level of protein sequence homology between species.

How do different CD9-FITC antibody clones compare in performance and specificity?

Different CD9-FITC antibody clones may exhibit significant variations in performance and specificity that can impact experimental outcomes. The MEM-61 clone (Mouse IgG1) has been validated for flow cytometry applications with human samples and cited in 4 publications . The EM-04 clone (Rat IgG1) is suitable for mouse samples in flow cytometry applications and has been cited in one publication . The 209306 clone has been validated for detecting CD9 in human platelets by flow cytometry . The ALB6 clone was first reported as specific for CD9 (p24) on platelets and capable of modulating aggregation of platelets activated with various agonists . The HI9a clone is also available but with less detailed performance information in the provided sources .

When selecting between these clones, researchers should consider the target species (human versus mouse), the specific epitope recognized, and published validation data. Cross-reactivity profiles differ significantly; for example, while some antibodies have been extensively tested in human samples (MEM-61, 209306, ALB6), others are specifically optimized for mouse samples (EM-04) . Epitope specificity also varies, with some antibodies recognizing extracellular domains while others may bind to different regions of the CD9 protein. This variability can be particularly important when studying CD9's multiple functional states or its interactions with other membrane proteins.

What are the optimal protocols for using CD9-FITC antibodies in multiparameter flow cytometry?

For multiparameter flow cytometry incorporating CD9-FITC antibodies, careful panel design is essential to minimize spectral overlap and maximize detection sensitivity. FITC has an emission peak at approximately 526 nm, which should be considered when selecting other fluorochromes to avoid substantial spillover . When designing panels that include CD9-FITC, consider pairing it with fluorochromes that have minimal spectral overlap with FITC, such as PE-Cy7, APC, or APC-Cy7.

The standard protocol involves adding 10 μl of CD9-FITC antibody per 10^6 cells in a 100 μl suspension or 10 μl per 100 μl of whole blood . Incubate the samples for 20-30 minutes at room temperature or 4°C in the dark. For whole blood samples, follow with an appropriate red blood cell lysis step using a commercial lysing solution according to the manufacturer's instructions. Wash cells twice with PBS containing 2% FBS to remove unbound antibody. If using other surface markers, it's generally safe to stain simultaneously with CD9-FITC, but validation is recommended.

Importantly, CD9 is sensitive to certain fixation methods, particularly those involving alcohols, which can alter the conformation of tetraspanin epitopes. If fixation is required, use a mild fixative such as 1-2% paraformaldehyde. Always include appropriate compensation controls when performing multiparameter flow cytometry, and be aware that the brightness of CD9-FITC may vary depending on the level of CD9 expression on your cells of interest.

How does CD9 expression correlate with cellular function in different research models?

CD9 expression levels correlate with several functional states across different cell types and research models. In oocytes, CD9 plays a crucial role in fertilization, organizing the membrane structure necessary for sperm-egg fusion . Knockout studies have shown that CD9-deficient female mice are infertile due to the inability of their eggs to fuse with sperm, highlighting the essential role of CD9 in reproductive biology .

In myoblasts, CD9 expression influences muscle regeneration by inhibiting myotube fusion. It accomplishes this by forming complexes with CD81 and PTGFRN . Expression of CD9 enhances membrane fusion between muscle cells and supports myotube maintenance, suggesting a dual regulatory role depending on the stage of muscle development .

In macrophages, CD9 expression regulates inflammatory responses, particularly in the lung, by modulating integrin-dependent migration . CD9 also prevents the fusion of macrophages into multinucleated giant cells and inhibits the formation of osteoclasts from mononuclear cell progenitors . This regulation of cell fusion events appears to be a common theme across different cellular contexts.

In cancer research models, CD9 expression has been linked to tumor metastasis and cell motility . Changes in CD9 expression levels may serve as prognostic markers in certain cancer types, though the relationship is complex and sometimes contradictory across different tumor types.

What are the technical considerations for detecting CD9 on extracellular vesicles using FITC-conjugated antibodies?

For flow cytometric detection of CD9 on EVs, specialized approaches are required. One effective method involves coupling EVs to larger beads (typically 4-5 μm aldehyde/sulfate latex or magnetic beads) to bring them into the detectable range. After bead coupling, CD9-FITC antibodies can be used to stain the EV-bound beads following a protocol similar to cell staining but with longer incubation times (typically 30-60 minutes).

The signal intensity from CD9-FITC on EVs will generally be lower than on cells due to the smaller surface area and potentially fewer CD9 molecules per vesicle. To enhance detection sensitivity, consider using antibody clones with higher affinity or brightness. Additionally, because exosomes contain multiple tetraspanins (CD9, CD63, CD81), co-staining with antibodies against these other markers can provide more comprehensive EV characterization.

A significant technical consideration is the purity of the EV preparation, as contaminating proteins or vesicles can lead to false positive signals. Always incorporate appropriate negative controls, such as isotype controls and beads without coupled EVs, to establish accurate detection thresholds. For quantitative applications, consider using calibration beads with known quantities of fluorophore to standardize fluorescence intensity measurements across experiments.

How can CD9-FITC antibodies be used to study tetraspanin-enriched microdomains?

CD9-FITC antibodies provide valuable tools for investigating tetraspanin-enriched microdomains (TEMs), which are specialized membrane domains where tetraspanins like CD9 interact with various partner proteins. To effectively study TEMs using CD9-FITC antibodies, researchers can employ several complementary approaches.

Flow cytometry with CD9-FITC provides quantitative assessment of CD9 expression levels across different cell populations but offers limited insight into the spatial organization of TEMs. For this reason, flow cytometry is often combined with microscopy techniques. Standard confocal microscopy using CD9-FITC antibodies can visualize the distribution of CD9 on the cell surface, while super-resolution techniques such as STORM or PALM can resolve individual TEMs at the nanoscale.

To analyze the protein composition of TEMs, co-immunoprecipitation experiments can be performed using CD9 antibodies (though unconjugated versions are typically used for this purpose), followed by proteomic analysis to identify interacting partners. Flow cytometry can then be used with CD9-FITC and antibodies against identified partners to quantify their co-expression across different cell types or experimental conditions.

When interpreting data from CD9-FITC staining, it's important to consider that antibody binding may itself influence TEM organization or signaling. Some antibody clones can induce clustering of CD9 or alter its interactions with partner proteins. Researchers should therefore validate findings using multiple approaches, including genetic manipulation of CD9 expression through knockdown or overexpression systems.

What are the best approaches for analyzing CD9 in platelets activation studies?

CD9 plays an important role in platelet activation and aggregation, making CD9-FITC antibodies valuable tools for studying platelet biology. Several approaches can be employed for analyzing CD9 in platelet activation studies.

Flow cytometry with CD9-FITC antibodies allows quantification of CD9 expression changes during platelet activation. The ALB6 clone has been specifically reported to modulate aggregation of platelets activated with various agonists, suggesting its utility in platelet functional studies . For flow cytometry analysis of platelets, careful sample preparation is crucial. Platelets should be isolated from fresh whole blood using gentle centrifugation techniques to avoid activation. Alternatively, CD9-FITC can be used to stain platelets directly in diluted whole blood followed by analysis of the platelet population based on forward and side scatter properties.

The table below summarizes a recommended protocol for analyzing CD9 in platelet activation studies:

How can CD9-FITC antibodies be used to investigate cellular fusion events?

CD9 plays critical regulatory roles in various cellular fusion events, including sperm-egg fusion, myoblast fusion, and macrophage fusion. CD9-FITC antibodies can be employed to investigate these processes through several experimental approaches.

For studying sperm-egg fusion, CD9-FITC can be used to quantify CD9 levels on oocytes by flow cytometry or visualize its distribution by microscopy. The functional importance of CD9 in this process can be assessed by using blocking antibodies against CD9 in fertilization assays. While FITC-conjugated antibodies are primarily used for detection rather than blocking, they can help confirm target engagement in blocking experiments.

In myoblast fusion studies, CD9-FITC antibodies allow tracking of CD9 expression changes during differentiation and fusion. Flow cytometry can quantify CD9 levels at different stages of myoblast differentiation, while time-lapse microscopy with CD9-FITC can visualize CD9 dynamics during fusion events. Since CD9 expression enhances membrane fusion between muscle cells and supports myotube maintenance , correlating CD9 levels with fusion efficiency can provide insights into the regulatory mechanisms.

For macrophage fusion investigations, CD9-FITC can monitor expression during conditions that promote fusion, such as exposure to IL-4 or foreign bodies. Since CD9 prevents macrophage fusion into multinucleated giant cells , decreased CD9 expression might predict increased fusion propensity. Flow cytometry with CD9-FITC can be combined with cell fusion assays to correlate CD9 levels with fusion outcomes.

When interpreting data from these studies, it's important to consider that CD9 functions within a network of tetraspanins and associated proteins. Therefore, analyzing CD9 in isolation may provide only partial insights into fusion regulation. Combining CD9-FITC with antibodies against other fusion-related proteins (CD81, integrins) can provide a more comprehensive understanding of the molecular mechanisms controlling cellular fusion events.

What are common issues with CD9-FITC antibody staining and how can they be resolved?

Researchers using CD9-FITC antibodies may encounter several common issues that can affect staining quality and experimental reproducibility. This section addresses these challenges and provides practical solutions.

Low signal intensity can occur due to several factors. If CD9 expression is naturally low on your cell type of interest, consider using more sensitive detection systems or amplification methods. For flow cytometry applications, ensure your instrument is properly calibrated and FITC voltage settings are optimized. Check antibody storage conditions, as FITC is sensitive to light exposure and may degrade over time. Store antibodies at 2-8°C protected from light, and avoid repeated freeze-thaw cycles .

High background staining may result from non-specific binding. Include proper blocking steps using serum (5-10%) from the same species as the secondary antibody (if applicable) or BSA (1-3%). Ensure thorough washing steps after antibody incubation, typically two washes with excess buffer. Always include appropriate isotype controls matched to your CD9-FITC antibody's host species and isotype .

Variable staining between experiments can be addressed through standardized protocols and consistent antibody lots. Use calibration beads to standardize instrument settings between experiments. Prepare single-stained controls for each experiment to verify antibody performance. Consider creating an antibody master mix for large experiments to ensure consistent antibody concentration across all samples.

If certain cell types show unexpected staining patterns, verify CD9 expression using alternative detection methods or antibody clones. Some cell treatments or fixation methods may alter CD9 epitopes. If working with fixed samples, optimize fixation conditions specifically for CD9 detection, as overfixation can mask epitopes while insufficient fixation may alter membrane protein distribution.

How can CD9-FITC antibody performance be optimized for different experimental conditions?

Optimizing CD9-FITC antibody performance requires tailoring protocols to specific experimental conditions. This section provides guidance for maximizing antibody performance across different applications and sample types.

For flow cytometry applications, antibody titration is essential for determining the optimal concentration that provides the best signal-to-noise ratio. Prepare a dilution series of CD9-FITC antibody (e.g., 0.25-10 μg/ml) and plot the signal-to-noise ratio against antibody concentration. The recommended starting point is 10 μl per 10^6 cells in 100 μl suspension, but optimal concentration may vary by cell type and antibody clone .

Sample preparation techniques should be optimized based on sample type. For blood samples, commercial lysing solutions are recommended for RBC removal after antibody staining . For adherent cells, gentle enzymatic dissociation methods (e.g., EDTA rather than trypsin) help preserve surface epitopes. For tissue samples, optimization of dissociation protocols is crucial for maintaining CD9 epitope integrity.

Buffer composition can be modified to enhance staining. Standard staining buffer contains PBS with 1-2% protein (BSA or FBS) and 0.1% sodium azide. For samples with high levels of Fc receptors (e.g., macrophages), adding an Fc blocking reagent before antibody staining reduces non-specific binding. For samples with high autofluorescence, including quenching agents or using specialized buffers may improve signal-to-noise ratio.

Multiplexed staining requires careful panel design. When combining CD9-FITC with other antibodies, consider the relative abundance of each marker and assign brighter fluorochromes to less abundant targets. FITC has moderate brightness, so CD9-FITC may not be ideal for detecting low-level CD9 expression in multiplexed panels. In such cases, consider using brighter fluorochromes conjugated to CD9 antibodies if available.