CD24 Antibody, FITC conjugated

What is CD24 and which cell types express this surface marker?

CD24 is a glycophosphatidylinositol (GPI)-anchored glycoprotein with a molecular weight ranging from 35-70 kDa, depending on its glycosylation pattern which varies by cell type. It is expressed on multiple cell populations including:

• B lymphocytes (from pro-B-cell stage through mature B cells, but not plasma cells)

• Granulocytes and neutrophils

• Eosinophils and dendritic cells

• Neural cells and epithelial cells

• Various cancer cells, particularly in B-lineage acute lymphoblastic leukemias

• Early thymocyte development stages

The protein functions as a signaling molecule that may be triggered through lectin-like ligand binding to its carbohydrate structures. CD24 modulates B-cell activation responses and, in conjunction with SIGLEC10, participates in selective suppression of immune responses to danger-associated molecular patterns (DAMPs) such as HMGB1, HSP70, and HSP90 .

What are the key differences between various clones of CD24-FITC antibodies?

Different monoclonal CD24-FITC antibodies exhibit distinct properties regarding species reactivity and epitope recognition:

When selecting a clone, researchers should consider both the target species and the specific epitope region required for their experimental design. For human samples, SN3 and ML5 are commonly employed, while mouse-focused research typically utilizes the 30-F1 clone .

How does FITC conjugation affect CD24 antibody functionality compared to other conjugates?

FITC (Fluorescein isothiocyanate) conjugation provides several advantages for flow cytometry applications:

• FITC demonstrates relatively high absorptivity with an excitation maximum around 495 nm

• It offers excellent fluorescence quantum yield with emission maximum at approximately 524 nm

• The conjugate maintains good water solubility, facilitating aqueous buffer usage

What is the optimal sample preparation protocol for CD24-FITC antibody staining in flow cytometry?

For optimal CD24-FITC staining in flow cytometry, follow this methodological approach:

• Cell preparation:

  • For peripheral blood: Use 100 μL of whole blood per test

  • For cell suspensions: Prepare approximately 1×10^6 cells per test

  • Wash cells twice with phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA)

• Antibody staining:

  • Add 20 μL of CD24-FITC antibody per 100 μL of whole blood or 10^6 cells

  • For mouse splenocytes, use 1 μg of antibody per 10^6 cells

  • Incubate for 30 minutes at 2-8°C in the dark

  • Wash twice with PBS/1% BSA to remove unbound antibody

• Red blood cell lysis (for whole blood):

  • Add 2 mL of lysing solution and incubate for 10 minutes

  • Centrifuge at 300-400×g for 5 minutes and discard supernatant

  • Wash cells twice with PBS/1% BSA

• Analysis:

  • Resuspend cells in 0.5 mL of PBS/1% BSA or appropriate fixative

  • Analyze on flow cytometer using appropriate instrument settings for FITC detection

  • Include appropriate isotype controls at the same concentration

This protocol minimizes non-specific binding while maintaining cellular viability and antigen integrity for accurate assessment of CD24 expression.

How should researchers design co-staining panels incorporating CD24-FITC for complex immunophenotyping?

When designing multi-parameter flow cytometry panels with CD24-FITC:

• Spectral considerations:

  • FITC emits in the green spectrum (524 nm), so avoid or compensate for fluorophores with substantial spectral overlap

  • Ideal companion fluorophores include PE (yellow), APC (red), and Pacific Blue (blue)

• Panel design strategy:

  • Assign CD24-FITC to targets of intermediate expression level

  • Reserve brighter fluorophores (PE, APC) for weakly expressed antigens

  • Use tandem dyes for strongly expressed markers

• B-cell lineage multi-parameter panel example:

  Marker	Fluorochrome	Purpose
  CD24	FITC	B-cell development stages
  CD19	PE	B-cell identification
  CD38	APC	Differentiation status
  CD45	Pacific Blue	Leukocyte common antigen

• Validation approach:

  • Perform fluorescence-minus-one (FMO) controls to establish proper gating

  • Include matched isotype controls for each fluorophore

  • Test compensation using single-stained controls

  • Verify with reference samples of known expression patterns

For effective co-staining, titrate each antibody separately before combining them to determine optimal concentrations that minimize background while maximizing signal-to-noise ratio. When analyzing mouse splenocytes, CD24-FITC paired with CD19-PE provides excellent discrimination of B-cell subpopulations as demonstrated in published research studies .

What are the critical quality control parameters for validating CD24-FITC antibody performance?

To ensure reliable experimental results with CD24-FITC antibodies, implement these quality control measures:

• Antibody validation tests:

  • Positive control testing: Verify staining on cell populations known to express CD24 (e.g., B lymphocytes, granulocytes)

  • Negative control testing: Confirm lack of binding to CD24-negative populations (e.g., plasma cells, certain T cell subsets)

  • Isotype control comparison: Use matched isotype-FITC at equivalent concentration to assess non-specific binding

  • Blocking experiment: Pre-incubate cells with unconjugated antibody to confirm specificity

• Instrument validation:

  • Fluorescence standardization using calibration beads

  • Consistent PMT voltage settings across experiments

  • Regular assessment of laser alignment and detector sensitivity

• Experimental controls:

  • Unstained samples to establish autofluorescence baseline

  • Single-stained compensation controls for multicolor experiments

  • Biological reference standards with known CD24 expression profiles

• Performance metrics:

  Parameter	Acceptable Range	Method of Assessment
  Signal-to-noise ratio	>20:1	Comparison of positive to negative populations
  Stain Index	>50	(MFI pos - MFI neg)/2×SD of negative population
  Lot-to-lot consistency	<10% variation	Comparative analysis of sequential lots
  Specificity	>95% agreement	Blocking studies and comparison to reference methods

• Storage and handling validation:

  • Antibody stability testing after storage at 2-8°C

  • Assessment of performance after exposure to room temperature

  • Protection from light exposure testing

Regular implementation of these quality control measures ensures consistent and reliable results across experimental timeframes.

How should researchers interpret variations in CD24 expression intensity across different cell populations?

Variations in CD24 expression intensity provide important biological information, but require careful interpretation:

• Normal variation patterns:

  • B-cell development stages show differential CD24 expression, with highest levels in pre-B and immature B cells, decreasing in mature B cells, and absent in plasma cells

  • Granulocyte populations typically display high CD24 expression with characteristic intensity

  • Epithelial cells may show intermediate expression levels

  • Neural cells demonstrate variable expression based on differentiation stage

• Interpretive framework:

  CD24 Expression Level	MFI Range	Typical Cell Types	Biological Significance
  High	>1000	Early B-cells, Granulocytes	Active developmental processes
  Intermediate	300-1000	Mature B-cells, Some epithelial cells	Functional signaling capacity
  Low	50-300	Transitional cells, Some T-cell subsets	Limited functional role
  Negative	<50	Plasma cells, Most T-cells	Terminal differentiation or lineage exclusion

• Methodological considerations affecting interpretation:

  • Antibody binding may be affected by glycosylation differences in CD24 between cell types

  • Clone-specific epitope availability can influence apparent expression levels

  • Cell preparation techniques (enzymatic digestion, fixation) may alter CD24 epitope accessibility

• Contextual analysis approach:

  • Always interpret CD24 expression in conjunction with other lineage and activation markers

  • Compare expression to established reference ranges for specific cell types

  • Consider physiological and pathological contexts when interpreting unexpected patterns

When analyzing expression patterns outside expected ranges, consider technical variables before attributing to biological significance, and validate with alternative detection methods when possible.

What are common technical challenges with CD24-FITC staining and their solutions?

Researchers frequently encounter technical issues with CD24-FITC antibody staining that can be systematically addressed:

• Poor signal intensity:

  • Cause: Insufficient antibody concentration, degraded antibody, or low target expression

  • Solution: Titrate antibody for optimal concentration; verify antibody stability with positive controls; extend incubation time to 45 minutes; ensure protection from light during all steps

• High background staining:

  • Cause: Non-specific binding, insufficient washing, Fc receptor interactions

  • Solution: Add Fc receptor blocking reagent prior to staining; increase wash cycles; use fresh buffer with protein carrier; verify isotype control performance

• Inconsistent staining across samples:

  Problem	Potential Cause	Corrective Action
  Variable population separation	Inconsistent sample processing	Standardize time from collection to staining
  Signal drift across samples	Photodegradation of FITC	Protect from light; process in batches
  Inter-assay variation	Temperature fluctuations	Maintain consistent temperature during incubation
  Unexpected negative results	Epitope masking or modulation	Try alternative clone or gentle fixation method

• Flow cytometer-related issues:

  • Cause: Suboptimal instrument settings, poor compensation, fluidics problems

  • Solution: Establish optimized FITC detector voltage; perform proper compensation with single-stained controls; ensure clean fluidics system; use quality control beads to monitor instrument performance

• Sample-specific challenges:

  • For whole blood: Incomplete red cell lysis causing high background

  • For tissue-derived cells: Digestion enzymes may cleave CD24 epitopes

  • For fixed samples: Overfixation may reduce antibody accessibility

When troubleshooting, systematically isolate variables by comparing to known positive controls and implementing changes individually to identify the specific cause of technical problems.

How can researchers differentiate between specific and non-specific binding when using CD24-FITC antibodies?

Distinguishing specific from non-specific binding is crucial for accurate data interpretation:

• Control implementation strategy:

  • Isotype control: Use fluorophore-matched isotype control antibody at identical concentration

  • Blocking experiment: Pre-incubate with excess unconjugated anti-CD24 before adding CD24-FITC

  • FMO control: Include all antibodies in panel except CD24-FITC to establish background

  • Known negative population: Identify CD24-negative populations (e.g., T-cells) as internal controls

• Analytical approaches:

  • Calculate signal-to-noise ratio between positive and negative populations

  • Compare staining pattern to established expression profiles for cell types

  • Evaluate staining intensity distribution (true positive binding typically shows distinct population shifts)

  • Assess concordance with alternative detection methods (e.g., different clones or techniques)

• Optimization tactics for reducing non-specific binding:

  Source of Non-specific Binding	Mitigation Strategy	Expected Improvement
  Fc receptor interaction	Add Fc block before antibody	70-90% reduction in background
  Dead cell binding	Include viability dye	Elimination of false positives
  Protein-protein interactions	Increase protein in buffer	Moderate reduction in background
  Charge-based binding	Adjust salt concentration	Variable improvement based on cause

• Quantitative assessment:

  • Calculate staining index: (MFI positive - MFI negative)/(2 × SD of negative)

  • Values >50 typically indicate specific binding

  • Examine fluorescence ratio between test sample and matched isotype control

  • Ratios >5 generally suggest specific binding

For definitive validation of questionable results, researchers should consider alternative detection methods or secondary confirmation approaches such as genetic knockdown of CD24 in relevant model systems.

How can CD24-FITC antibodies be applied in cancer stem cell identification and characterization?

CD24 expression patterns serve as critical markers for cancer stem cell (CSC) identification across multiple tumor types:

• Cancer-specific CD24 expression patterns:

  • Breast cancer: CD24-/low phenotype often identifies cancer stem cells when combined with CD44+

  • Pancreatic cancer: CD24+ cells in combination with CD44+ and ESA+ mark tumorigenic populations

  • Colorectal cancer: Variable CD24 expression depending on CSC subpopulation

  • Hepatocellular carcinoma: CD24+ cells demonstrate enhanced self-renewal and tumorigenicity

• Methodological approach for CSC identification:

  • Multi-parameter flow cytometry combining CD24-FITC with:

    • CD44-PE for breast and pancreatic cancer stem cells

    • EpCAM/ESA-APC for epithelial tumors

    • CD133-PE or CD133-APC for various solid tumors

  • Isolation of CD24+ or CD24- populations via FACS for functional assays

  • Xenograft studies to confirm tumorigenic capacity of sorted populations

• Functional characterization procedures:

  Assay Type	Methodology	CD24-Related Outcome
  Sphere formation	Low-attachment culture	CD24-/low cells form more numerous/larger spheres in breast cancer
  Chemoresistance	Drug exposure followed by viability assessment	CD24+ cells show enhanced survival in some cancers
  Invasion capacity	Transwell migration assays	CD24 expression correlates with invasive capacity in pancreatic cancer
  In vivo tumorigenicity	Limited dilution xenograft	CD24+/CD44+ cells initiate tumors at lower cell numbers

• CD24-targeted therapeutic applications:

  • Antibody-drug conjugates targeting CD24+ cancer cells

  • CAR-T cell therapy directed against CD24-expressing tumors

  • Monitoring treatment response through CD24+ circulating tumor cell quantification

When designing CSC studies using CD24-FITC, researchers should include comprehensive functional validation beyond simple phenotypic characterization, as CD24 expression patterns alone may not definitively identify CSCs across all tumor types and contexts.

What is the role of CD24 in autoimmunity research and how can CD24-FITC antibodies contribute to this field?

CD24 plays significant regulatory roles in autoimmune processes, making CD24-FITC antibodies valuable research tools:

• CD24 functions in autoimmunity regulation:

  • Modulates B-cell activation responses crucial in autoantibody production

  • In association with Siglec-10/G, selectively suppresses immune responses to danger-associated molecular patterns (DAMPs)

  • Influences T-cell homeostasis and activation thresholds

  • Regulates dendritic cell function and inflammatory cytokine production

• Experimental applications of CD24-FITC in autoimmunity research:

  • Quantitative assessment of CD24 expression across immune cell subsets in autoimmune conditions

  • Correlation of CD24 expression levels with disease severity and progression

  • Monitoring CD24+ B-cell populations during experimental therapeutic interventions

  • Isolation of CD24-defined cellular subsets for functional and transcriptomic characterization

• Methodological approaches:

  Research Question	Experimental Design	CD24-FITC Application
  CD24 polymorphism influence	Genotype-phenotype correlation	Flow cytometric quantification of expression levels
  Therapeutic response monitoring	Longitudinal patient sampling	Tracking CD24+ cell population dynamics
  Mechanistic investigation	CD24 knockout models	Validation of CD24 absence in experimental animals
  B-cell tolerance mechanisms	Antigen-specific B-cell analysis	Co-staining with autoantigen tetramers and CD24-FITC

• Disease-specific research focuses:

  • Multiple sclerosis: CD24 expression on pathogenic T-cells and its correlation with disease progression

  • Systemic lupus erythematosus: CD24 levels on autoreactive B-cells

  • Rheumatoid arthritis: CD24 expression patterns in synovial infiltrating lymphocytes

  • Type 1 diabetes: Role of CD24 in regulating diabetogenic T-cell responses

Researchers investigating autoimmunity should consider implementing longitudinal CD24 expression analysis in patient cohorts, correlating with clinical parameters and other immunological markers to establish CD24's predictive and mechanistic relevance in specific autoimmune conditions.

How can CD24-FITC antibodies be implemented in developmental and stem cell biology studies?

CD24-FITC antibodies provide valuable tools for tracking developmental processes and stem cell differentiation:

• Developmental biology applications:

  • Neural development: CD24 marks differentiating neuroblasts and neural progenitor cells

  • B-lymphocyte development: CD24 expression changes mark distinct developmental stages

  • Epithelial morphogenesis: CD24 expression patterns correlate with differentiation states

• Stem cell characterization and isolation:

  • Hematopoietic stem/progenitor cell identification in combination with other markers

  • Neural stem cell population purification and characterization

  • Tracking differentiation trajectories of pluripotent stem cells

• Experimental design strategies:

  Research Application	Methodology	Key Considerations
  Lineage tracing	Flow cytometric monitoring of CD24 during differentiation	Temporal sampling at critical developmental timepoints
  Progenitor isolation	FACS sorting based on CD24-FITC and complementary markers	Optimization of antibody concentration for FACS
  Differentiation potential assessment	Colony-forming assays with sorted CD24+ vs CD24- cells	Functional validation of sorted populations
  In vivo developmental tracking	Analysis of CD24 expression in tissue sections or dissociated cells	Combined flow cytometry and immunohistochemistry approaches

• Optimized multi-parameter panel for stem cell studies:

  • CD24-FITC: Developmental stage marker

  • CD44-PE: Stem cell adhesion molecule

  • CD49f-APC: Progenitor marker

  • Lin markers-Pacific Blue: Lineage exclusion

  • Viability dye-Far Red: Dead cell discrimination

• Analytical considerations:

  • Correlation of CD24 expression levels with functional stem cell properties

  • Single-cell analysis techniques to resolve heterogeneous expression patterns

  • Integration with transcriptomic profiling of CD24-defined populations

When implementing CD24-FITC in developmental studies, researchers should establish clear baseline expression patterns for their specific model system, as CD24 expression dynamics vary significantly between tissue types and developmental contexts.

How does CD24 antibody performance compare across different detection platforms beyond flow cytometry?

While CD24-FITC antibodies are optimized for flow cytometry, CD24 detection extends to multiple platforms with varying considerations:

• Cross-platform comparison:

  Platform	Antibody Format	Sensitivity	Resolution	Key Advantages	Limitations
  Flow cytometry	FITC conjugated	High	Single-cell	Quantitative, multi-parameter	Requires cell suspensions
  Immunohistochemistry	Unconjugated primary	Moderate	Tissue context	Spatial information, morphology	Semi-quantitative
  Immunofluorescence	FITC or unconjugated	Moderate-high	Subcellular	Localization, co-localization	Photo-bleaching concerns
  Western blotting	Unconjugated primary	Low-moderate	Population	Size verification	Glycosylation variability
  CyTOF/Mass cytometry	Metal-conjugated	High	Single-cell	No compensation needed	Specialized equipment

• Epitope accessibility considerations:

  • Formaldehyde fixation may mask certain CD24 epitopes in tissue sections

  • Clone-specific differences in performance across platforms (e.g., SN3 vs. ML5)

  • Glycosylation-dependent epitopes may be affected by sample processing

• Optimization strategies for non-flow applications:

  • For IHC/IF: Antigen retrieval methods must be optimized for CD24

  • For Western blotting: Sample denaturation affects epitope recognition

  • For frozen tissue sections: Clone SN3 and ML5 have demonstrated reliability

• Novel and emerging platforms:

  • Imaging mass cytometry for high-dimensional tissue analysis

  • Single-cell RNA-seq correlation with protein expression

  • Spatial transcriptomics coupled with CD24 immunofluorescence

Researchers should validate CD24 antibody performance on their specific detection platform rather than assuming transferability from flow cytometry applications, particularly given the complex glycosylation of CD24 that affects epitope accessibility.

What are the advanced analytical approaches for interpreting complex CD24 expression patterns in heterogeneous samples?

Modern analytical tools enhance the interpretation of complex CD24 expression data:

• Computational flow cytometry approaches:

  • Unsupervised clustering algorithms (e.g., FlowSOM, PhenoGraph)

  • Dimensionality reduction techniques (tSNE, UMAP) for visualizing CD24 expression in high-dimensional space

  • Trajectory inference methods to map developmental progressions based on CD24 and companion markers

• Quantitative analysis framework:

  Analytical Method	Application	Advantage for CD24 Analysis
  Density-based clustering	Identification of rare CD24+ subpopulations	Robust to noise and outliers
  Earth Mover's Distance	Comparing CD24 distributions between samples	Quantifies distribution shifts
  Jensen-Shannon Divergence	Measuring changes in CD24 expression	Statistical rigor for heterogeneity assessment
  Mixture modeling	Decomposing complex CD24 expression patterns	Identifies overlapping populations

• Integration with multi-omics data:

  • Correlation of CD24 protein expression with single-cell transcriptomics

  • CITE-seq approaches combining CD24-antibody detection with RNA profiling

  • Integration of CD24 flow cytometry data with epigenetic profiling

  • Functional network analysis incorporating CD24 expression data

• Machine learning applications:

  • Predictive modeling of functional properties based on CD24 expression patterns

  • Classification algorithms for disease state prediction using CD24 and companion markers

  • Deep learning approaches for automated identification of CD24-defined cell populations

When applying these advanced analytical techniques, researchers should implement appropriate validation approaches, including manual gating verification, biological plausibility assessment, and cross-validation across independent samples to ensure robust interpretation of CD24 expression data.

How can researchers design controlled experiments to elucidate CD24 functional mechanisms in complex biological systems?

Rigorous experimental design is essential for investigating CD24's functional roles:

• Genetic manipulation approaches:

  • CRISPR/Cas9-mediated CD24 knockout to assess loss-of-function effects

  • Inducible CD24 expression systems to study gain-of-function

  • Site-directed mutagenesis of CD24 glycosylation sites to determine structure-function relationships

  • Knockin of reporter genes (e.g., fluorescent proteins) to track CD24 expression dynamics

• Functional assay framework:

  Investigation Focus	Experimental Approach	Readout Method	CD24-FITC Role
  Signaling mechanism	Biochemical pathway inhibition	Phospho-flow cytometry	Cell identification
  Adhesion function	Ligand blocking studies	Adhesion/migration assays	Population isolation
  Immune regulation	Mixed lymphocyte reactions	Proliferation assays	Cell sorting
  Development	Lineage-specific CD24 conditional knockout	Phenotypic analysis	Population tracking

• Controlled experimental variables:

  • Use of matched genetic backgrounds for comparisons

  • Implementation of littermate controls in animal studies

  • Inclusion of both positive controls (known CD24 functions) and negative controls

  • Dose-response studies for antibody-mediated functional modulation

• Advanced techniques for mechanism elucidation:

  • Proximity ligation assays to identify CD24 interaction partners

  • Super-resolution microscopy to visualize CD24 membrane distribution

  • CD24 immunoprecipitation followed by mass spectrometry

  • Single-molecule tracking to assess CD24 dynamics in living cells