CD45 Antibody, FITC

What is CD45 and why is it significant in immunological research?

CD45, also known as protein tyrosine phosphatase receptor type C, is a type I transmembrane protein expressed on all hematopoietic cells with the exception of erythrocytes and platelets. The protein serves as a pan-hematopoietic cell marker and plays a crucial role in immune cell function. CD45 is essential for both T- and B-cell antigen receptor-mediated activation and signaling, making it a fundamental molecule in adaptive immunity. Alternative names include leukocyte common antigen (LCA), T200, and Ly5, reflecting its discovery history across different research groups. The protein exists in multiple isoforms (180, 190, 205, 220 kDa) generated through alternative splicing of three exons, which contributes to its diverse functions in different cell populations .

What are the optimal handling and storage conditions for CD45-FITC antibodies?

CD45-FITC antibodies require specific storage conditions to maintain their functionality and fluorescence properties. These conjugated antibodies should be stored at 2-8°C and protected from light exposure, which can degrade the fluorescent properties of FITC. Most commercially available CD45-FITC antibodies come in liquid form in phosphate-buffered saline (PBS) containing 0.09-0.1% sodium azide and may include 0.5% bovine serum albumin (BSA) as a stabilizer. When stored under optimal conditions, these antibodies typically remain stable for one year after shipment. It's important to avoid repeated freeze-thaw cycles, as this can compromise antibody integrity and reduce binding efficiency .

What cell types express CD45 and can be detected using CD45-FITC antibodies?

CD45 expression serves as a reliable marker for cells of hematopoietic origin. CD45-FITC antibodies can detect expression on:

• T lymphocytes

• B lymphocytes

• Monocytes

• Macrophages

• Natural killer (NK) cells

• Granulocytes

• Dendritic cells

Notably, CD45 is absent on erythrocytes and platelets, making it useful for distinguishing nucleated blood cells from these elements. The HI30 antibody clone in particular is widely used as a marker for human CD45 expression across the full spectrum of leukocytes . In experimental settings, CD45-FITC antibodies have been validated using human peripheral blood mononuclear cells (PBMCs), demonstrating their reliability in identifying CD45-positive populations for immunological research .

What are the standard dilutions and applications for CD45-FITC antibodies?

CD45-FITC antibodies are versatile reagents that can be employed in multiple experimental techniques. The recommended dilutions vary by application:

For flow cytometry applications specifically, many commercial antibodies are pre-titrated, with a standard recommendation of 5 μl per 10^6 cells in a 100 μl suspension, or 5 μl per 100 μl of whole blood .

How can CD45-FITC antibodies be used to determine antigen saturation in different tissues?

Antigen saturation analysis using CD45-FITC antibodies provides critical information about antibody binding efficiency and CD45 expression levels across different tissue compartments. The methodology involves a comparative approach using two different staining strategies:

• Primary detection method: Cells are first incubated with unlabeled anti-CD45 antibody, followed by staining with FITC-conjugated goat anti-mouse F(ab')2 (FMF) to detect bound primary antibody.

• Direct detection method: Cells are stained directly with FITC-conjugated anti-CD45 antibody (CD45F).

By comparing the mean fluorescence intensity (MFI) between these two methods, researchers can determine the degree of CD45 saturation. When most CD45 sites are occupied by unlabeled antibody, the FMF signal will be high while the CD45F signal will be low, indicating near-complete saturation.

Research data demonstrates tissue-specific differences in CD45 targeting efficiency. For example, in one study, bone marrow lymphocytes showed rapid and efficient targeting by anti-CD45 antibodies, with high FMF intensity and low CD45F signals at early time points. In contrast, lymph node samples exhibited low FMF staining regardless of time point or antibody dose, with corresponding high CD45F intensity, indicating abundant free antigen .

The following data exemplifies this methodology:

This approach is particularly valuable for radioimmunotherapy applications where understanding the biodistribution and binding efficiency of anti-CD45 antibodies across different tissue compartments is essential for predicting therapeutic efficacy .

What are the critical considerations for using CD45-FITC in multicolor flow cytometry panels?

Incorporating CD45-FITC into multicolor flow cytometry panels requires careful consideration of several technical factors to ensure accurate and interpretable results:

• Spectral overlap management: FITC has excitation/emission maxima at approximately 495nm/524nm. When designing multicolor panels, researchers must account for potential spectral overlap with other fluorochromes like PE, which may necessitate compensation adjustments. The specific FITC conjugation used in CD45 antibodies has reported excitation/emission maxima of 495 nm/524 nm .

• Clone selection relevance: Different anti-CD45 clones may have varying epitope specificities and binding characteristics. For human samples, commonly used clones include 2D1 and HI30, each with established performance characteristics. The choice of clone should be consistent with research objectives and validated for the specific cell populations under investigation .

• Fixation compatibility: When protocols require cell fixation and permeabilization, it's important to verify that the CD45-FITC antibody maintains its reactivity. Some antibodies are specifically validated for use with fixed/permeabilized cells, with adjusted dilution recommendations (typically 1:20) .

• Gating strategy optimization: As a pan-leukocyte marker, CD45 is often used for initial identification and gating of total leukocyte populations. In bone marrow or peripheral blood analyses, CD45 expression intensity can help distinguish between different hematopoietic cell lineages when combined with side scatter properties.

• Antibody titration: Despite manufacturer recommendations, optimal antibody concentration should be determined through titration experiments for each specific application and sample type, particularly for rare cell populations or when sample preparation methods differ from standard protocols.

How can CD45-FITC be used in targeting strategies for hematopoietic cell transplantation research?

CD45-FITC antibodies play a vital role in research focused on hematopoietic cell transplantation (HCT) targeting strategies, particularly in the development of radioimmunotherapy approaches that may substitute for total body irradiation in preparative regimens. The methodology incorporates CD45-FITC in several critical capacities:

• Target validation: CD45-FITC antibodies can confirm the expression and accessibility of CD45 on target cell populations prior to therapeutic intervention. This validation step ensures that the therapeutic antibody will effectively reach the intended targets.

• Biodistribution assessment: By using CD45-FITC in parallel with experimental therapeutic antibodies, researchers can track the tissue-specific distribution of anti-CD45 binding. Flow cytometry analysis of cell suspensions from blood, lymph nodes, and bone marrow aspirates provides quantitative data on antibody penetration and binding efficiency across different compartments.

• Saturation analysis: As detailed previously, comparing the mean fluorescence intensity of cells incubated with either FITC-conjugated secondary antibodies (detecting bound unlabeled anti-CD45) or directly with CD45-FITC allows researchers to determine the degree of antigen saturation in different tissues.

• Treatment efficacy monitoring: Flow cytometry dot plots of FITC versus forward scatter can indicate cell depletion in specific compartments following treatment, providing a readout of therapeutic efficacy. For example, research has shown cell depletion in bone marrow at later time points after anti-CD45 radioimmunotherapy, while similar effects were not observed in lymph nodes .

• Dose optimization: The relationship between antibody dose and CD45 saturation can be quantified using CD45-FITC, informing the development of dosing strategies for therapeutic applications. Studies have examined different antibody doses (e.g., 0.75 mg/kg vs. 1.00 mg/kg) to determine optimal targeting parameters .

This methodological approach has supported the development of alpha-radioimmunotherapy targeting CD45 as a potential substitute for total body irradiation in HCT preparative regimens for conditions like lymphoma .

What are the optimal protocols for using CD45-FITC in different microscopy applications?

CD45-FITC antibodies can be effectively employed across various microscopy techniques, each requiring specific protocol optimization:

• Immunofluorescence (Immunocytochemistry):

  • Recommended dilution range: 1:50 - 1:200, with exact dilution requiring optimization for each application

  • Cell fixation: 4% paraformaldehyde (10-15 minutes) followed by permeabilization with 0.1-0.5% Triton X-100 if intracellular epitopes are targeted

  • Blocking: 5-10% normal serum (species-dependent on secondary antibody) with 1% BSA

  • Primary incubation: Directly apply diluted CD45-FITC antibody, incubate 1-2 hours at room temperature or overnight at 4°C

  • Counterstaining: DAPI for nuclear visualization

  • Mounting: Anti-fade mounting medium to preserve fluorescence

• Immunohistochemistry (Frozen Sections):

  • Tissue preparation: Flash freezing followed by cryosectioning (5-10 μm)

  • Fixation: Cold acetone (10 minutes)

  • Blocking: 10% normal serum with 1% BSA

  • Antibody application: Similar to immunocytochemistry

  • For dual labeling: A reliable method involves combining immunogold-silver staining with immunoenzymatic labeling for simultaneous demonstration of two antigens

• Immunohistochemistry (Paraffin Sections):

  • Deparaffinization: Standard xylene and alcohol series

  • Antigen retrieval: Critical step, typically heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Blocking: Endogenous peroxidase block (3% H₂O₂) followed by protein block

  • Primary antibody: CD45-FITC applied at optimized dilution

  • Visualization: Anti-FITC secondary antibody followed by chromogenic or fluorescent detection systems

• Advanced co-localization studies:

  • For simultaneous demonstration of two antigens, researchers have successfully employed a combination of immunogold-silver staining and immunoenzymatic labeling techniques

  • This approach allows for precise spatial relationship analysis between CD45 and other markers of interest

Each application requires careful optimization and validation, with particular attention to fixation conditions that preserve both epitope accessibility and FITC fluorescence properties.

How do different clones of CD45-FITC antibodies compare in terms of specificity and sensitivity?

Different clones of CD45-FITC antibodies exhibit varying characteristics that can significantly impact experimental outcomes in terms of specificity, sensitivity, and application compatibility. Understanding these differences is crucial for selecting the most appropriate reagent for specific research questions:

• Clone 2D1 (Southern Biotech):

  • Immunogen: Human peripheral blood mononuclear cells

  • Isotype: Mouse (BALB/c) IgG₁κ

  • Applications: Validated for flow cytometry, immunohistochemistry (frozen and paraffin sections), and western blot (non-reducing conditions)

  • Special features: Recognizes all isoforms of CD45 (180, 190, 205, 220 kDa)

  • Cross-reactivity: Specific for human CD45 with no reported cross-reactivity to other species

• Clone HI30 (Cell Signaling Technology):

  • Isotype: Mouse IgG₁

  • Applications: Optimized for immunofluorescence and flow cytometry (both live and fixed/permeabilized cells)

  • Dilution requirements: 1:20 for flow cytometry, 1:50-1:200 for immunofluorescence

  • Target specificity: Widely used as a marker for human CD45 expression on all leukocytes including T cells, B cells, monocytes, macrophages, and NK cells

  • Sensitivity: Detects endogenous levels of CD45 protein

• Proteintech antibody (FITC-65109):

  • Host/Isotype: Mouse IgG₁, kappa

  • Immunogen: Human peripheral blood leukocytes

  • Purification method: Affinity purification

  • Applications: Pre-titrated specifically for flow cytometric analysis

  • Usage recommendation: 5 μl per 10^6 cells in a 100 μl suspension or 5 μl per 100 μl of whole blood

  • FITC characteristics: Excitation/Emission Maxima Wavelengths: 495 nm/524 nm

Comparative evaluation factors to consider:

• Epitope specificity: Different clones may bind to distinct epitopes on the CD45 molecule, potentially affecting detection of specific isoforms

• Background staining: Some clones may exhibit lower non-specific binding, particularly important in tissues with high autofluorescence

• Stability after fixation: Certain clones maintain better reactivity following various fixation protocols

• Compatibility with multicolor panels: Performance in the context of other fluorochromes may vary between clones

When selecting between these clones, researchers should consider their specific application requirements, the nature of their samples, and whether prior literature has established precedent for using particular clones in their research area .

What are the best practices for optimizing CD45-FITC staining in flow cytometry?

Optimizing CD45-FITC staining for flow cytometry requires attention to several methodological details to ensure reliable and reproducible results:

• Antibody titration: Despite manufacturer recommendations, performing a titration experiment is essential to determine the optimal antibody concentration for your specific cell type and experimental conditions. Create a dilution series (typically 2-fold) and plot signal-to-noise ratio against antibody concentration to identify the optimal dilution that maximizes specific staining while minimizing background.

• Sample preparation considerations:

  • Fresh vs. frozen samples: CD45 epitopes generally maintain stability during freezing, but validation is recommended when transitioning between fresh and frozen protocols

  • Red blood cell lysis: When working with whole blood, use gentle lysis buffers that preserve leukocyte viability and surface marker expression

  • Buffer composition: PBS with 0.5-2% BSA or FBS helps reduce non-specific binding

  • Fc receptor blocking: Include an Fc blocking step (10-15 minutes at 4°C) before antibody addition to reduce non-specific binding, particularly for samples containing B cells or monocytes/macrophages

• Staining protocol optimization:

  • Temperature: While room temperature staining is common, some epitopes demonstrate better binding at 4°C

  • Incubation time: Standard protocols suggest 20-30 minutes, but extending to 45-60 minutes may improve signal for certain applications

  • Washing steps: Insufficient washing can lead to high background, while excessive washing may reduce signal intensity

• Instrument settings:

  • PMT voltage: Adjust to position the negative population in the first decade of the logarithmic scale

  • Compensation: Critical when using FITC in multicolor panels, particularly with PE due to spectral overlap

  • Threshold/trigger: Setting appropriate thresholds on forward scatter or CD45 can help eliminate debris and non-target events

• Controls implementation:

  • Fluorescence minus one (FMO) controls: Essential for accurate gating, especially in multicolor panels

  • Isotype controls: Mouse IgG1-FITC (such as clone 15H6) serves as an appropriate isotype control

  • Biological controls: Include known positive and negative samples to validate staining patterns

For quantitative applications, standardization using calibration beads with known numbers of molecules of equivalent soluble fluorochrome (MESF) can convert fluorescence intensity to absolute values, enabling comparison across experiments and instruments .

How can researchers effectively validate CD45-FITC antibody performance in their specific experimental systems?

Rigorous validation of CD45-FITC antibodies is essential to ensure reliable experimental outcomes. A comprehensive validation strategy includes:

• Positive and negative control samples:

  • Positive controls: Human peripheral blood mononuclear cells (PBMCs) serve as reliable positive controls for CD45 expression

  • Negative controls: Erythrocytes and platelets should show no CD45 staining, providing internal negative controls in blood samples

  • Cell lines: Well-characterized cell lines with known CD45 expression profiles (e.g., Jurkat for T cells, Raji for B cells) can serve as standardized controls

• Antibody specificity confirmation:

  • Western blot analysis: Can confirm binding to proteins of expected molecular weights (180-220 kDa depending on isoform)

  • Competitive binding assays: Pre-incubation with unlabeled anti-CD45 should block subsequent CD45-FITC binding

  • Knockout/knockdown models: Where available, CD45-deficient cells provide definitive negative controls

• Functional validation approaches:

  • Correlation with biological function: CD45 expression should correlate with expected functional attributes of specific leukocyte populations

  • Activation studies: Changes in CD45 isoform expression following cell activation can be monitored to confirm antibody specificity

  • Cross-platform validation: Concordance between flow cytometry results and immunohistochemistry findings strengthens validation

• Technical performance assessment:

  • Titration analysis: Signal-to-noise ratio optimization through systematic dilution series

  • Stability testing: Evaluate performance after various storage conditions and durations

  • Lot-to-lot consistency: Comparison between different manufacturing lots using standardized samples

  • Fixation compatibility: Test performance with different fixation and permeabilization protocols relevant to your research

• Data analysis validation:

  • Gating strategy reproducibility: Develop and test gating approaches using multiple samples and operators

  • Quantitative linearity: For quantitative applications, verify linear relationship between antibody concentration and signal intensity

  • Precision assessment: Replicate measurements to determine intra- and inter-assay coefficients of variation

Implementing this systematic validation approach ensures that CD45-FITC antibody performance is optimized for specific experimental conditions and provides confidence in the reliability and reproducibility of research findings .

What approaches can address common challenges in CD45-FITC antibody applications?

Researchers frequently encounter several challenges when working with CD45-FITC antibodies. Here are methodological solutions to address these issues:

• High background fluorescence:

  • Challenge: Non-specific binding or autofluorescence interfering with specific CD45 signal

  • Solutions:

    • Implement stringent blocking (10% serum from the same species as secondary antibody plus 1% BSA)

    • Include 0.1-0.3% Triton X-100 in blocking buffer to reduce hydrophobic interactions

    • Increase washing steps (3-5 washes with PBS + 0.05% Tween-20)

    • When working with tissues with high autofluorescence (e.g., brain, lung), consider using Sudan Black B (0.1-0.3%) treatment post-staining

    • Consider alternative fluorophores with emission spectra outside the autofluorescence range for highly autofluorescent samples

• Weak or variable staining intensity:

  • Challenge: Insufficient signal strength or inconsistent staining patterns

  • Solutions:

    • Optimize antibody concentration through careful titration experiments

    • Extend incubation time (overnight at 4°C instead of 1-2 hours at room temperature)

    • Verify buffer pH (optimal range typically 7.2-7.4)

    • Enhance epitope accessibility through optimized antigen retrieval methods

    • For fixed samples, test multiple fixation protocols to identify optimal conditions

    • Consider signal amplification systems for low-abundance targets

• Loss of FITC fluorescence intensity:

  • Challenge: FITC is relatively prone to photobleaching

  • Solutions:

    • Minimize exposure to light during all steps of the protocol

    • Use anti-fade mounting media containing radical scavengers

    • Store slides at -20°C if they need to be archived

    • Consider rapid image acquisition protocols to minimize exposure time

    • If repeated imaging is necessary, consider more photostable alternatives to FITC

• Cross-reactivity concerns:

  • Challenge: Potential binding to non-target molecules

  • Solutions:

    • Validate antibody specificity using knockout/knockdown controls where available

    • Perform competitive binding assays with unlabeled antibody

    • Include appropriate isotype controls (Mouse IgG1-FITC for most CD45-FITC antibodies)

    • When analyzing complex tissues, include single-color controls to verify specificity

• Sample-specific optimization requirements:

  • Challenge: Protocols optimized for one sample type may not translate to others

  • Solutions:

    • For bone marrow samples: Adjust for higher cell density and consider lineage-specific gating strategies

    • For lymph node samples: Optimize tissue disaggregation protocols to maintain epitope integrity

    • For whole blood: Implement gentle RBC lysis protocols to preserve leukocyte viability

    • For paraffin-embedded tissues: Optimize antigen retrieval (heat-induced epitope retrieval with citrate buffer pH 6.0 or EDTA buffer pH 9.0)

    • For frozen sections: Test different fixation protocols (acetone, paraformaldehyde, or methanol) to identify optimal conditions

By systematically addressing these challenges with appropriate methodological adjustments, researchers can significantly improve the reliability and reproducibility of CD45-FITC antibody applications across diverse experimental contexts .