ITGAM Monoclonal Antibody,FITC Conjugated

What is ITGAM/CD11b and what biological functions does it serve?

CD11b (integrin alpha-M, ITGAM) is a 165-170 kDa adhesion molecule that non-covalently associates with integrin beta-2 (CD18) to form the Mac-1 complex (αMβ2 integrin, CR3). This heterodimeric complex plays crucial roles in various adhesive interactions of immune cells and in mediating the uptake of complement-coated particles and pathogens. CD11b/CD18 functions as a receptor for multiple ligands including ICAM-1 (CD54), ICAM-2 (CD102), iC3b, fibrinogen, and factor X, facilitating critical cell-cell and cell-matrix interactions. In the immune system, CD11b participates in neutrophil migration, phagocytosis, and inflammatory responses. Recent studies have also linked genetic variants in ITGAM to systemic lupus erythematosus (SLE), highlighting its importance in autoimmune pathology .

What cell types express CD11b/ITGAM and how can this be utilized in research?

CD11b is predominantly expressed on the surface of myeloid lineage cells, including monocytes/macrophages, granulocytes (particularly neutrophils), and microglia in the brain. Additionally, it is found on activated lymphocytes, a subset of natural killer (NK) cells, and certain dendritic cell populations. The M1/70 antibody clone is widely used as a marker for CD11b expression on these cell types. This expression pattern makes CD11b antibodies valuable tools for identifying and sorting specific immune cell populations in both human and mouse samples, particularly in studies of inflammation, infection, and immune cell trafficking. The differential expression of CD11b on various cell types allows researchers to distinguish between myeloid and lymphoid populations in complex tissues and blood samples .

How do FITC-conjugated anti-CD11b antibodies work in flow cytometry?

FITC (Fluorescein isothiocyanate)-conjugated anti-CD11b antibodies bind specifically to CD11b molecules on the cell surface. When excited by a blue laser (typically 488 nm) during flow cytometry, the FITC fluorophore emits light at approximately 520 nm, allowing for detection of CD11b-positive cells. The intensity of this fluorescence signal correlates with the level of CD11b expression on the cell surface. For optimal flow cytometry results, manufacturers typically recommend using 0.5 μg of antibody per test, with a test defined as the amount of antibody needed to stain a cell sample in a final volume of 100 μL. Cell numbers can range from 10^5 to 10^8 cells per test, though researchers should empirically determine the optimal cell concentration and antibody titration for their specific experimental conditions .

How should I optimize anti-CD11b-FITC antibody staining protocols for flow cytometry?

Optimizing anti-CD11b-FITC antibody staining requires careful consideration of several variables. Begin with antibody titration to determine the optimal concentration that maximizes signal-to-noise ratio. Though manufacturers typically recommend a 1:100 dilution or 0.5 μg per test, optimal concentrations may vary based on your specific cell population and experimental conditions. For live cell staining, use cold PBS containing 2% FBS and 0.1% sodium azide as your staining buffer, and maintain cells at 4°C throughout the procedure to prevent antibody internalization and preserve cell viability. For fixed/permeabilized cells, ensure your fixation protocol doesn't affect the CD11b epitope recognized by your specific antibody clone (M1/70 or ICRF44). When designing multi-color panels, be aware of potential spectral overlap between FITC and other fluorophores like PE or GFP, and include appropriate single-stain controls for compensation. Finally, always include unstained controls, isotype controls (Rat IgG2b kappa for M1/70 or Mouse IgG1 for ICRF44), and fluorescence-minus-one (FMO) controls to accurately set gates for CD11b-positive populations .

What are the key differences between the M1/70 and ICRF44 anti-CD11b antibody clones?

The M1/70 and ICRF44 clones represent two distinct monoclonal antibodies targeting CD11b/ITGAM with important differences in species reactivity, epitope recognition, and application versatility. The M1/70 clone is a rat IgG2b kappa antibody that demonstrates cross-reactivity with both mouse and human CD11b, making it particularly valuable for comparative studies or researchers working with both species. In contrast, the ICRF44 clone is a mouse monoclonal antibody specifically targeting human CD11b with no reported cross-reactivity to mouse samples. Regarding epitope recognition, while both antibodies bind CD11b, they likely recognize different epitopes, which may affect their binding in certain experimental conditions where protein conformation is altered. The M1/70 clone has been extensively validated for flow cytometry applications in both fixed/permeabilized and live cells, while ICRF44 is primarily recommended for flow cytometry of human samples. Researchers should select the appropriate clone based on their target species, required applications, and whether cross-reactivity is desirable for their experimental design .

How can I effectively use CD11b-FITC antibodies in studies of neutrophil function?

For effective use of CD11b-FITC antibodies in neutrophil function studies, begin by optimizing your neutrophil isolation protocol to minimize activation, as mechanical stress can upregulate CD11b surface expression and confound results. Consider using density gradient separation with Polymorphprep or magnetic negative selection for high purity. When designing experiments to assess neutrophil activation, remember that CD11b expression increases rapidly upon stimulation with inflammatory mediators, making it an excellent marker for early activation. To study phagocytic capacity, combine CD11b-FITC staining with phagocytosis assays using complement-coated erythrocytes or serum-treated zymosan particles, which specifically engage the Mac-1 complex. For adhesion studies, pre-stain neutrophils with CD11b-FITC before adhesion assays to ICAM-1-coated surfaces or endothelial monolayers. In genetic variation studies, particularly those examining ITGAM variants associated with SLE, use CD11b-FITC to assess whether these variants alter the surface expression of Mac-1 in addition to functional assays examining phagocytosis and adhesion. Finally, when studying neutrophil extracellular trap (NET) formation, CD11b staining can help identify the activation state of neutrophils prior to NETosis .

How do ITGAM genetic variants affect Mac-1 function and how can this be assessed with FITC-conjugated antibodies?

ITGAM genetic variants, particularly the non-synonymous SNPs rs1143679, rs1143678, and rs1143683 associated with systemic lupus erythematosus (SLE), can significantly alter Mac-1 function on neutrophils and other myeloid cells. These functional alterations can be comprehensively assessed using FITC-conjugated anti-CD11b antibodies in several complementary approaches. First, flow cytometric analysis using CD11b-FITC allows quantification of basal and stimulation-induced surface expression levels of Mac-1, which may be altered in cells carrying risk variants. Second, combining CD11b-FITC staining with phagocytosis assays using complement-coated erythrocytes, serum-treated zymosan, or heat-treated zymosan provides direct measurement of CR3-mediated phagocytic capacity. The mean fluorescence intensity of CD11b-FITC can be correlated with phagocytic index to determine if reduced function correlates with altered expression. Third, adhesion assays to ICAM-1, fibrinogen, or iC3b-coated surfaces can reveal defects in Mac-1-dependent cell adhesion, which can be confirmed through blocking experiments with unlabeled anti-CD11b antibodies. Finally, calcium flux assays combined with CD11b-FITC staining can assess whether ITGAM variants affect Mac-1-mediated signaling. When designing these experiments, researchers should genotype and sequence donors for all known ITGAM variants to ensure comprehensive analysis of genetic effects on Mac-1 function .

What are the considerations for multiplexed analysis of CD11b with other myeloid markers?

Multiplexed analysis of CD11b with other myeloid markers requires careful panel design to maximize information while minimizing fluorescence spillover artifacts. When designing a multicolor panel including CD11b-FITC, consider the brightness hierarchy of your markers and fluorophores. FITC has moderate brightness, so pair CD11b-FITC with abundant antigens while reserving brighter fluorophores (PE, APC) for lower-expressed markers. Position FITC in your panel to minimize spillover into adjacent channels, particularly PE and PerCP-Cy5.5. For comprehensive myeloid cell phenotyping, combine CD11b-FITC with markers that define specific subpopulations: Ly6G for neutrophils (in mouse), CD14/CD16 for monocyte subsets, HLA-DR for activation status, and CD15 for granulocytes (in human samples). When examining tissue-resident macrophages, include F4/80 (mouse) or CD68 (human) alongside CD11b to distinguish between resident and infiltrating populations. For functional studies, combine CD11b-FITC with markers of activation (CD66b, CD62L) and intracellular markers of effector function (myeloperoxidase, lactoferrin) using appropriate fixation and permeabilization protocols. Always perform proper compensation controls and consider using specialized dimensionality reduction analysis methods like tSNE or UMAP to fully resolve complex myeloid subpopulations in your data .

How can CD11b-FITC antibodies be used to study neutrophil extracellular traps (NETs) formation?

CD11b-FITC antibodies serve as valuable tools in the multifaceted study of neutrophil extracellular trap (NET) formation through several methodological approaches. For live-cell imaging of NETosis progression, CD11b-FITC can be used to track the redistribution of this receptor during the process, as CD11b undergoes significant membrane reorganization prior to NET release. This can be combined with DNA stains like Hoechst or DAPI and additional markers such as myeloperoxidase or neutrophil elastase. For quantitative flow cytometry analysis, researchers can stimulate neutrophils with PMA, microbial stimuli, or autoantibodies, then measure CD11b upregulation as an early activation marker that precedes NET formation. CD11b expression levels can be correlated with other NET markers to establish temporal relationships in the NETosis pathway. Additionally, CD11b-FITC antibodies can be used to sort CD11b-high and CD11b-low neutrophil populations prior to NET induction assays to determine if CD11b expression levels predict NET-forming capacity. When studying the role of Mac-1 in NETosis directly, researchers can use blocking experiments with unlabeled anti-CD11b antibodies prior to stimulation, then compare NET formation using quantitative imaging or DNA release assays. Importantly, when using anti-CD11b antibodies in NET studies, researchers should ensure the staining protocol doesn't inadvertently activate neutrophils, potentially using Fc-blocking reagents and maintaining samples at 4°C during antibody incubation .

What are common issues with CD11b-FITC staining and how can they be resolved?

Common issues with CD11b-FITC staining include several technical challenges that can be systematically addressed. First, if experiencing high background fluorescence, implement more rigorous blocking steps using 10% normal serum from the same species as your secondary antibody (if applicable) or use commercial Fc receptor blocking reagents. Additionally, include a viability dye to exclude dead cells which often bind antibodies non-specifically. For weak or variable CD11b staining, consider that CD11b expression can be rapidly upregulated during mechanical manipulation of cells—maintain strict sample handling protocols, keep cells at 4°C, and include sodium azide in staining buffers to prevent modulation of surface expression. If detecting abnormal CD11b downregulation, check if your samples were inadvertently exposed to activation stimuli like bacterial products or inflammatory cytokines, which can trigger CD11b internalization in certain conditions. For inconsistent results between experiments, standardize your gating strategy using fluorescence-minus-one (FMO) controls and beads for voltage standardization across flow cytometry sessions. When working with fixed/permeabilized samples, some fixatives can affect the CD11b epitope—test multiple fixation protocols with your specific antibody clone. Finally, if using samples from patients on anti-integrin therapies, be aware that therapeutic antibodies may compete with your detection antibody for epitope binding .

How should CD11b-FITC antibodies be stored and handled to maintain optimal performance?

Proper storage and handling of CD11b-FITC antibodies is critical for maintaining their performance characteristics over time. FITC-conjugated antibodies are particularly sensitive to light exposure, which can cause photobleaching and reduced fluorescence intensity. Store the antibody in opaque containers or wrap standard vials in aluminum foil, and minimize light exposure during experimental procedures. Temperature management is equally important—store antibodies at 2-8°C for short-term (1-2 weeks) usage, but for long-term storage, aliquot the antibody into small volumes and keep at -20°C to avoid repeated freeze-thaw cycles, which can cause protein denaturation and aggregation. When preparing working dilutions, use high-quality buffers containing appropriate stabilizers (BSA or serum) and preservatives (sodium azide at 0.02-0.1%). Never vortex antibody solutions vigorously as this can cause protein denaturation; instead, mix by gentle inversion or flicking. Prior to each use, centrifuge antibody vials briefly (10,000g for 20 seconds) to collect liquid at the bottom and remove any precipitates. Always check the manufacturer's expiration date and lot-specific quality control data for each antibody. When designed experiments involve quantitative comparisons of CD11b expression levels across multiple experiments, consider using the same lot of antibody throughout the study to avoid lot-to-lot variations in fluorochrome-to-protein ratios that could affect staining intensity .

What are the optimal instrument settings for detecting FITC-conjugated antibodies in flow cytometry?

Optimizing instrument settings for FITC-conjugated antibody detection requires attention to several technical parameters. FITC is optimally excited by the blue 488 nm laser and emits fluorescence with a peak at approximately 520 nm. Configure your flow cytometer's filter sets to capture this emission, typically using a 530/30 nm bandpass filter. Begin setup by running unstained cells to adjust forward scatter (FSC) and side scatter (SSC) voltages to appropriately display your cell population of interest. For FITC voltage adjustment, run a single-stained FITC-positive control and adjust the detector voltage so that the positive population falls within the upper log decade of the scale without reaching the maximum. Be aware that FITC has significant spectral overlap with PE (phycoerythrin), which must be compensated for in multicolor panels. Set compensation using single-stained controls with the same antibody concentrations used in your experiment. For instruments with automated setup features, use FITC-labeled calibration beads to standardize target fluorescence values across experiments. When analyzing samples with variable CD11b expression levels, ensure your dynamic range is sufficient by including both high and low expressers in your panel design. For precise quantification of CD11b expression, consider using commercially available calibration beads with known quantities of FITC molecules to convert fluorescence intensity to antibodies bound per cell (ABC). Finally, record sufficient events (minimum 10,000 for rare populations) to ensure statistical robustness in your CD11b expression analysis .

How should researchers interpret changes in CD11b expression levels in different experimental conditions?

Interpreting changes in CD11b expression requires careful consideration of biological context and technical variables. CD11b is a dynamic marker that rapidly upregulates upon cellular activation, making it valuable for monitoring neutrophil and monocyte activation states. When analyzing CD11b expression data, first establish baseline expression in resting cells and standardize analysis by reporting fold-change in mean fluorescence intensity (MFI) relative to this baseline rather than absolute values. Consider the biphasic nature of CD11b regulation—initial stimulation causes rapid translocation of preformed CD11b from intracellular granules to the cell surface (occurring within minutes), while sustained activation induces de novo synthesis (occurring over hours). Time-course experiments can distinguish between these phases. Be aware that CD11b can be shed from the cell surface under certain inflammatory conditions, potentially resulting in reduced detection despite cellular activation. For studies comparing expression between different donors or genotypes (such as ITGAM variants in SLE), normalize data to account for individual variations in baseline expression. When analyzing tissue-derived samples, consider that enzymatic digestion methods may cleave CD11b or alter epitope accessibility, potentially causing artificially low staining. Finally, integrate CD11b expression data with functional assays, as expression levels may not always directly correlate with functional capacity due to conformational changes that affect ligand binding independently of expression level .

What is the significance of CD11b expression heterogeneity within cell populations?

CD11b expression heterogeneity within cell populations represents a biologically significant phenomenon that may reflect functional diversity, developmental stages, or activation states. When analyzing flow cytometry data, resist the temptation to simply gate on "CD11b-positive" versus "CD11b-negative" populations, as the distribution is often continuous rather than bimodal. Instead, consider subdividing myeloid populations into CD11b-low, CD11b-intermediate, and CD11b-high subsets, analyzing each separately for additional phenotypic and functional markers. In neutrophils, CD11b expression heterogeneity may identify developmental subsets, with immature neutrophils generally expressing lower CD11b levels than mature cells. During acute inflammation, CD11b-high neutrophils typically represent activated cells with enhanced effector functions. In monocytes, CD11b expression varies among classical (CD14++CD16-), intermediate (CD14++CD16+), and non-classical (CD14+CD16++) subsets, providing additional resolution of monocyte heterogeneity. Utilize dimensionality reduction techniques such as tSNE, UMAP, or FlowSOM to visualize how CD11b expression correlates with other markers across the entire myeloid compartment. Perform cell sorting of different CD11b-expressing subsets followed by functional assays (phagocytosis, ROS production, cytokine secretion) or transcriptomic analysis to determine if expression heterogeneity correlates with functional specialization. Finally, in longitudinal studies, track how the proportions of these CD11b-defined subsets change during disease progression or in response to therapeutic interventions .

How can CD11b-FITC data be integrated with other datasets for comprehensive immune profiling?

Integrating CD11b-FITC flow cytometry data with other datasets enables comprehensive immune profiling that transcends the limitations of any single analytical approach. For multi-omic integration, begin by designing experiments with matched samples for flow cytometry, transcriptomics, and functional assays. Cell sorting based on CD11b expression levels followed by RNA-seq or proteomics can reveal molecular signatures associated with different CD11b-defined myeloid subsets. When combining with functional data, correlate CD11b expression with functional readouts like phagocytic capacity, respiratory burst activity, or cytokine production on a per-cell basis using imaging flow cytometry or flow cytometry with functional reporters. For clinical studies, integrate CD11b expression data with patient metadata, including disease activity scores, treatment response, and long-term outcomes to identify biomarker potential. Computational integration requires careful attention to data normalization—consider using approaches like z-scoring or quantile normalization when comparing flow cytometry data across multiple time points or patient cohorts. Employ machine learning approaches such as random forest algorithms or support vector machines to identify complex relationships between CD11b expression patterns and other immune parameters that may not be apparent with conventional statistical methods. Network analysis can position CD11b+ cells within the broader immune ecosystem by examining correlations between CD11b-expressing populations and other immune cell types across samples. Finally, validate key findings from integrative analyses using orthogonal methods—for instance, if transcriptomic data suggests a novel function for CD11b-high cells, confirm this with targeted functional assays .

What is the role of CD11b in disease pathogenesis and how can FITC-conjugated antibodies help study this?

CD11b plays multifaceted roles in disease pathogenesis across various conditions, which can be effectively studied using FITC-conjugated antibodies. In autoimmune diseases like systemic lupus erythematosus (SLE), genetic variants in ITGAM are associated with disease susceptibility, with certain variants altering neutrophil function by impairing phagocytosis and adhesion. CD11b-FITC antibodies enable researchers to correlate variant-specific expression levels with functional defects in neutrophils from genotyped individuals, providing mechanistic insights into how these variants contribute to pathogenesis. In inflammatory conditions, CD11b mediates neutrophil adhesion to endothelium and subsequent extravasation into inflamed tissues—processes that can be tracked by flow cytometric analysis of CD11b upregulation following inflammatory stimuli. In neurodegenerative diseases, CD11b expressed on microglia contributes to neuroinflammation through production of superoxide ions, promoting neuronal apoptosis. Flow cytometric analysis of microglial CD11b expression in animal models can reveal activation states associated with neurodegeneration. For infectious diseases, CD11b functions as a pattern recognition receptor that binds various microbial components, facilitating phagocytosis and clearance—processes that can be quantified using CD11b-FITC in combination with labeled pathogens. In cardiovascular disease, CD11b+ monocytes contribute to atherosclerotic plaque formation, with CD11b-FITC enabling researchers to track monocyte subsets associated with disease progression. By providing quantitative, cell-specific data on CD11b expression and correlating this with functional outcomes, FITC-conjugated anti-CD11b antibodies serve as essential tools for elucidating the role of this integrin in diverse pathological processes .

What emerging technologies complement CD11b-FITC antibody staining for comprehensive myeloid cell analysis?

Several emerging technologies are complementing traditional CD11b-FITC antibody staining to enable more comprehensive myeloid cell analysis. Spectral flow cytometry with unmixing algorithms allows for the use of fluorophores with overlapping emission spectra, enabling higher-parameter analysis of CD11b+ cells alongside numerous additional markers. This technology can distinguish subtle myeloid subsets based on 30+ parameters simultaneously. Mass cytometry (CyTOF) uses metal-tagged antibodies rather than fluorophores, eliminating spectral overlap concerns and enabling 40+ parameter analysis of CD11b+ myeloid cells with exquisite subset resolution. Single-cell RNA sequencing coupled with protein expression (CITE-seq) combines transcriptomic profiling with antibody-based detection of surface proteins, allowing researchers to correlate CD11b protein expression with genome-wide transcriptional programs at single-cell resolution. For tissue-based analysis, multiplexed immunofluorescence techniques like Imaging Mass Cytometry or CODEX enable visualization of CD11b+ cells in their tissue context alongside dozens of other markers, preserving spatial relationships critical for understanding myeloid cell functions in complex tissues. Advanced computational tools including deep learning algorithms can identify novel myeloid cell clusters based on high-dimensional flow cytometry data that includes CD11b-FITC, discovering populations not evident with manual gating strategies. Intravital microscopy combined with photoactivatable fluorescent CD11b antibodies allows for real-time tracking of myeloid cell trafficking and behavior in live animals. Finally, engineered reporter mice expressing fluorescent proteins under the control of the ITGAM promoter enable longitudinal tracking of CD11b+ cells without requiring repeated antibody staining, particularly valuable for in vivo imaging studies .

What are the most significant recent findings regarding CD11b function that impact experimental design with FITC-conjugated antibodies?

Recent significant findings regarding CD11b function have important implications for experimental design using FITC-conjugated antibodies. First, researchers have discovered that CD11b exists in multiple conformational states with different ligand-binding affinities, transitioning between inactive and active conformations upon cellular activation. This finding necessitates the use of conformation-specific antibodies alongside traditional expression analysis—researchers should be aware whether their particular anti-CD11b-FITC clone (M1/70 or ICRF44) recognizes specific conformational states. Second, studies have revealed that CD11b undergoes post-translational modifications, including glycosylation patterns that vary between cell types and activation states, potentially affecting antibody binding. Researchers should validate their CD11b-FITC antibody's performance across different cell types and activation conditions. Third, new research demonstrates that CD11b signaling varies contextually depending on ligand engagement—binding to iC3b may trigger different downstream pathways than binding to ICAM-1 or fibrinogen. When studying specific CD11b functions, researchers should design experiments that selectively engage specific ligand-receptor interactions rather than relying solely on expression analysis. Fourth, recent studies highlight CD11b's role in regulating Toll-like receptor signaling and inflammasome activation, suggesting important immunomodulatory functions beyond adhesion and phagocytosis. Experimental designs should incorporate readouts for these pathways when studying CD11b+ cells. Fifth, the discovery that certain ITGAM variants associated with SLE specifically impair Mac-1 interactions with specific ligands while preserving others emphasizes the need for comprehensive functional testing rather than assuming uniform functional impairment based on expression levels alone. Finally, the identification of CD11b as a regulator of neutrophil extracellular trap (NET) formation opens new experimental applications—researchers studying NETosis should consider CD11b not just as a phenotypic marker but as a functional contributor to this process .

CD11b expression profile across immune cell populations

This table provides a comprehensive overview of CD11b expression across various immune cell populations, including relative expression levels and functional significance. This information is crucial for researchers designing flow cytometry panels for immune cell phenotyping and for interpreting CD11b expression data in different cellular contexts .