CD11b Antibody, FITC

What is CD11b and which cell populations express it?

CD11b (also known as integrin alpha-M or ITGAM) is a 165-170 kDa adhesion glycoprotein that non-covalently associates with CD18 (integrin beta-2) to form the alphaMbeta2 integrin complex, also known as Mac-1 or CR3 (complement receptor 3) . This heterodimeric complex is expressed on multiple immune cell populations including:

• Monocytes/macrophages

• Neutrophils and other granulocytes

• Activated lymphocytes

• A subset of natural killer (NK) cells

• Dendritic cells (DCs)

• Microglia in the brain

• Mouse B-1 cells in the peritoneal cavity

CD11b expression varies by tissue and activation state, with approximately 8% of spleen cells, 44% of bone marrow cells, and less than 1% of thymocytes expressing this marker . CD11b/CD18 serves as a receptor for multiple ligands including ICAM-1 (CD54), ICAM-2 (CD102), ICAM-4 (CD242), CD14, CD50, CD23, heparin, iC3b, fibrinogen, and Factor X, mediating critical cell-cell and cell-matrix interactions .

While manufacturers provide general recommendations, optimal antibody concentration should be empirically determined for each experimental system. The typical recommended concentrations are:

• For M1/70 clone: ≤0.5 μg per test or 1:100 dilution

• For ICRF44 clone: 5 μL (0.5 μg) per test

A titration experiment is strongly recommended to determine the optimal signal-to-noise ratio:

• Prepare a dilution series of the antibody (e.g., 2 μg, 1 μg, 0.5 μg, 0.25 μg, 0.125 μg per test)

• Stain identical aliquots of your target cells with each concentration

• Include appropriate isotype controls (e.g., Rat IgG2b kappa for M1/70, Mouse IgG1 kappa for ICRF44)

• Calculate the stain index for each concentration: (MFI positive - MFI negative) / (2 × SD of negative)

• Select the concentration that provides the highest stain index while minimizing background

The optimal antibody concentration may vary depending on the specific cell population, sample type (e.g., peripheral blood vs. tissue), and experimental conditions.

What are the spectral characteristics of FITC-conjugated CD11b antibodies?

FITC-conjugated CD11b antibodies have the following spectral properties:

• Excitation maximum: 488 nm

• Emission maximum: 520 nm

• Compatible with: Blue laser (typically 488 nm)

What are the recommended protocols for staining CD11b in fixed versus live cells?

The staining protocol differs significantly between fixed and live cells:

For Live Cell Staining:

• Harvest cells and resuspend in flow cytometry staining buffer (PBS with 0.5-2% BSA and 0.1% sodium azide)

• Add 0.5 μg (or manufacturer's recommended amount) of CD11b-FITC antibody per 10^6 cells

• Incubate for 30 minutes at 4°C protected from light

• Wash twice with staining buffer

• Analyze immediately or fix with 1-2% paraformaldehyde if analysis will be delayed

For Fixed/Permeabilized Cell Staining:

• Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

• Wash twice with PBS

• If intracellular staining is required, permeabilize with 0.1% saponin or commercial permeabilization buffer

• Add CD11b-FITC antibody at 1:100 dilution in permeabilization buffer

• Incubate for 45-60 minutes at room temperature protected from light

• Wash twice with permeabilization buffer followed by a final wash with staining buffer

• Analyze by flow cytometry

Researchers should note that fixation can alter CD11b epitopes, potentially affecting antibody binding. The M1/70 clone has shown good resistance to fixation effects, while other clones may exhibit reduced binding after fixation . Always include appropriate controls to validate staining in fixed samples.

How do I properly compensate for FITC in multicolor flow cytometry panels containing CD11b?

Proper compensation is critical for accurate analysis of multicolor panels containing CD11b-FITC:

• Prepare single-stained controls:

  • For each fluorochrome in your panel, prepare a sample stained with only that fluorochrome

  • For FITC compensation, use CD11b-FITC on the same cell type as your experiment

  • Alternatively, use compensation beads (e.g., BD CompBeads) following manufacturer's protocol

• Set up compensation matrix:

  • Adjust voltage settings to place negative populations in the first decade of the logarithmic scale

  • Ensure positive signals are on-scale but not at maximum intensity

  • Use automated compensation tools in your flow cytometry software

  • Review and manually adjust compensation if necessary

• Verify compensation accuracy:

  • Examine bivariate plots of FITC vs. each other fluorochrome

  • Properly compensated data should show aligned median fluorescence intensity for positive and negative populations

Note that BD CompBeads and similar products can have slightly different spectral properties compared to cells for some fluorochromes. It is recommended to compare spillover on cells and compensation beads to ensure appropriate compensation, especially when using CD11b-FITC in critical applications .

How can I validate the specificity of CD11b-FITC antibody staining in my experimental samples?

Validating antibody specificity is essential for reliable data interpretation:

• Isotype controls: Include an isotype-matched control antibody (Rat IgG2b kappa-FITC for M1/70, Mouse IgG1 kappa-FITC for ICRF44) to assess non-specific binding

• Biological controls:

  • Positive control: Samples known to express high levels of CD11b (e.g., mouse bone marrow cells, human peripheral blood monocytes)

  • Negative control: Samples with minimal CD11b expression (e.g., lymphocyte populations)

  • Visualize staining as shown in Figure 1 of search result , which demonstrates clear separation between positive and negative populations

• Blocking experiments:

  • Pre-incubate cells with unlabeled CD11b antibody before staining with CD11b-FITC

  • Specific staining should be significantly reduced

• Comparative analysis:

  • Compare staining patterns with multiple CD11b antibody clones

  • Results should be consistent across different clones targeting the same epitope

Careful validation ensures that observed signals genuinely represent CD11b expression rather than artifacts or non-specific binding.

How does CD11b expression change during monocyte maturation and activation?

CD11b expression is dynamically regulated during monocyte differentiation and activation:

• Monocyte maturation:

  • CD11b expression increases progressively during monocyte maturation

  • Mature monocytes express higher levels than immature precursors

  • Expression levels vary on tissue macrophages depending on tissue microenvironment

• Activation-induced changes:

  • Inflammatory stimuli (e.g., LPS, TNF-α) typically upregulate CD11b surface expression

  • This upregulation correlates with enhanced adhesive and migratory properties

  • Translocation of preformed intracellular CD11b to the cell surface occurs rapidly (within minutes)

  • De novo synthesis contributes to sustained elevation over hours

• Tissue-specific expression:

  • Peritoneal macrophages express higher CD11b levels than splenic macrophages

  • Microglia in the brain use CD11b as a characteristic marker

  • Regulation varies by tissue microenvironment factors

Researchers studying monocyte/macrophage biology should consider these dynamic changes when interpreting CD11b expression data, as expression levels reflect both developmental stage and activation status.

How can I use CD11b-FITC antibodies in combination with other markers to identify specific myeloid subpopulations?

Combining CD11b-FITC with additional markers enables precise identification of myeloid subpopulations:

For mouse samples:

For human samples:

When designing multicolor panels, consider the following:

• Place CD11b-FITC in a channel with minimal spillover from brighter markers

• Avoid using PE-conjugated antibodies for dim antigens when using FITC due to spillover challenges

• Include viability dye to exclude dead cells, which can bind antibodies non-specifically

• Establish gates using fluorescence-minus-one (FMO) controls

What are the potential pitfalls when interpreting CD11b expression data in inflammatory conditions?

Interpreting CD11b expression in inflammatory settings requires awareness of several potential confounding factors:

• Activation-induced shedding:

  • Under strong inflammatory stimuli, CD11b can be cleaved from the cell surface

  • This shedding may lead to underestimation of the actual inflammatory state

  • Consider measuring soluble CD11b in supernatants/plasma in conjunction with surface staining

• Post-translational modifications:

  • CD11b function is regulated by conformational changes and phosphorylation

  • These modifications may affect antibody binding without changing actual expression levels

  • Antibodies recognizing activation-dependent epitopes may show variable results

• Environmental influences:

  • Sample processing (temperature changes, mechanical stress) can artificially activate myeloid cells

  • This activation can increase CD11b surface expression independent of the biological condition

  • Standardize sample handling protocols and include appropriate controls

• Context-dependent expression patterns:

  • In some chronic inflammatory conditions, CD11b may be counterintuitively downregulated

  • Different inflammatory mediators can have opposing effects on CD11b expression

  • Always interpret CD11b data in context with other inflammatory markers

To mitigate these pitfalls, researchers should include time-course analyses, measure additional activation markers, and standardize sample collection and processing protocols.

How do I troubleshoot inconsistent CD11b staining in my experimental samples?

Inconsistent CD11b staining can arise from multiple sources:

• Antibody-related issues:

  • Fluorochrome degradation: FITC is particularly susceptible to photobleaching; store protected from light

  • Antibody aggregation: Centrifuge vial briefly before use to pellet any aggregates

  • Lot-to-lot variation: Validate new lots against previous ones using the same experimental system

• Sample preparation problems:

  • Delayed processing: Process samples immediately; CD11b expression changes ex vivo

  • Excessive mechanical manipulation: Gentle handling prevents artificial activation

  • Improper fixation: Some fixatives can damage CD11b epitopes; optimize fixation protocols

• Technical variability:

  • Inconsistent compensation: Re-establish compensation matrices for each experiment

  • Instrument variation: Regular quality control using standardized beads

  • Temperature fluctuations: Maintain consistent temperature during staining (typically 4°C for live cells)

• Biological factors:

  • Circadian rhythms: CD11b expression on neutrophils fluctuates throughout the day

  • Medication effects: Many drugs affect CD11b expression; document patient/animal treatments

  • Recent infections or inflammatory events: Even subclinical conditions can alter expression

Systematic troubleshooting approach:

• Run positive and negative control samples alongside experimental samples

• Use a secondary reporting marker to confirm cell identification

• Evaluate sample viability; dead cells often show non-specific binding

• Consider testing alternative CD11b antibody clones

What are the best practices for quantifying CD11b expression levels in flow cytometry?

Accurate quantification of CD11b expression requires rigorous methodology:

• Metrics for expression quantification:

  • Percentage of positive cells: Useful for bimodal expression patterns

  • Mean/median fluorescence intensity (MFI): Better for shifts in expression level

  • Molecules of equivalent soluble fluorochrome (MESF): Standardized units allowing cross-experiment comparison

  • Antibody binding capacity (ABC): Most accurate measure of actual receptor numbers

• Standardization approaches:

  • Include calibration beads with known quantities of fluorochrome

  • Use the same voltage settings and PMT gain across experiments

  • Include biological reference samples in each experiment

  • Apply standardized gating strategies using fixed gates or automated algorithms

• Advanced quantification techniques:

  • Quantitative flow cytometry using beads with defined ABC values

  • MESF calibration to convert raw fluorescence to standardized units

  • Digital processing with standardized transformation algorithms

• Documentation requirements:

  • Record instrument settings, voltage, and compensation matrices

  • Document lot numbers of antibodies and reagents

  • Maintain detailed protocols including incubation times and temperatures

  • Include all control samples used for standardization

For longitudinal studies or multi-center collaborations, implementing rigorous standardization protocols is essential for meaningful comparison of CD11b expression data.