CD21 Antibody, FITC

What is CD21 and what is its significance in immunological research?

CD21, also known as complement receptor type 2 (CR2), complement C3d receptor, and Epstein-Barr virus receptor, is a transmembrane protein critical for immunological studies. It contains a small cytoplasmic domain, a transmembrane region, and an extracellular domain composed of 15 tandem short consensus repeat sequences . CD21 plays multiple essential roles in the immune system:

• It functions as a receptor for complement fragments C3d, C3dg, and iC3b

• It serves as the primary receptor for Epstein-Barr virus (EBV)

• On B cells, CD21 forms part of the CD19/CD21/CD81 co-receptor complex associated with the B cell receptor (BCR)

• It is crucial for B cell activation and function

The importance of CD21 in research stems from its central role in immune regulation, particularly in B cell activation and immune complex processing. Studies utilizing CD21-deficient (Cr2null) mice have demonstrated impaired inflammatory and humoral immune responses, highlighting CD21's significance in adaptive immunity .

What is the expression pattern of CD21 across different cell types?

CD21 shows a distinct expression pattern across immune cell populations:

In B-cell ontogeny, CD21 appears after the pre-B-stage, is maintained during peripheral B-cell development, and is lost upon terminal differentiation into plasma cells. CD21 expression is also gradually lost after stimulation of B cells in vitro .

What are the optimal protocols for using CD21 Antibody, FITC in flow cytometry?

For optimal results when using CD21 Antibody, FITC in flow cytometry, follow these methodological guidelines:

Sample Preparation and Staining:

• Use 5 μl of antibody per 10^6 cells in a 100 μl suspension or 5 μl per 100 μl of whole blood

• Incubate samples according to standard flow cytometry protocols

• For mouse studies using the 7G6 clone: DO NOT fix cells after staining as this severely reduces staining intensity (even ≤1 hour with 1% formaldehyde can affect results)

Sample Handling Post-Staining:

• Freshly-isolated leukocytes and cell lines may be kept in wash buffer at 4°C without fixation for up to 18 hours post-staining without significant loss of viability

• For activated lymphocytes, which may lose viability more rapidly, collect data within 5 hours post-staining

Technical Considerations:

• Since applications may vary, each investigator should titrate the reagent to determine optimal concentration for their specific experimental system

• For comprehensive protocols, refer to manufacturer-specific technical resources

How does the CD21/CD19/CD81 complex function in B cell signaling?

The CD21/CD19/CD81 complex serves as a crucial co-receptor to the B cell receptor (BCR), significantly enhancing B cell activation and function through several mechanisms:

CD21 binds complement fragments C3d, C3dg, and iC3b that opsonize antigens, effectively lowering the threshold for B cell activation by connecting the innate and adaptive immune responses . This complex functions as a signal-transduction assembly that works synergistically with the BCR to amplify activation signals .

When antigens are bound by both the BCR and CD21 (through complement fragments), the signaling potency increases by 10-1000 fold compared to BCR engagement alone. The molecular basis for this includes:

• CD19 contains multiple tyrosine residues in its cytoplasmic domain that become phosphorylated upon complex activation

• Phosphorylated CD19 recruits signaling molecules including PI3K and Vav

• CD81 (TAPA-1) is a tetraspanin that helps organize the complex in the membrane

• Together, these components enhance calcium flux, activate MAP kinases, and promote B cell proliferation and differentiation

In knockout models, loss of CD21 (Cr2null mice) results in impaired humoral immune responses, particularly to T-dependent antigens, demonstrating the physiological importance of this complex in normal B cell function .

What are the specific characteristics of different CD21 antibody clones?

Different CD21 antibody clones exhibit distinct binding characteristics and applications:

The 7G6 clone is particularly notable for its ability to recognize both CD21 and CD35 in mouse samples. This antibody has been validated for use in various immunological assays, but researchers should note its sensitivity to fixation when FITC-conjugated .

The hCD21 clone specifically targets human CD21 and has been validated for flow cytometry applications. It is particularly useful for identifying CD21-expressing cells in human peripheral blood mononuclear cells (PBMCs) .

How does fixation affect FITC-conjugated CD21 antibody performance?

Fixation can significantly impact the performance of FITC-conjugated CD21 antibodies, with notable differences depending on the specific clone and experimental conditions:

For the 7G6 clone (mouse CD21/CD35), studies have demonstrated that fixation severely reduces staining intensity, even after short fixation periods (≤1 hour with 1% formaldehyde) . This phenomenon necessitates specific handling protocols:

• Researchers should avoid fixing cells prior to flow cytometry analysis

• Freshly-isolated leukocytes and cell lines can be maintained in wash buffer at 4°C without fixation for up to 18 hours post-staining

• For activated lymphocytes, which tend to lose viability more rapidly, data collection should occur within 5 hours of staining

The mechanism behind this fixation sensitivity likely involves conformational changes to the fluorochrome or epitope accessibility alterations that reduce fluorescence intensity. Alternative approaches include:

• Analyzing samples without fixation when possible

• Considering alternative conjugates if fixation is absolutely required

• Optimizing fixation protocols specifically for CD21 detection if necessary

What are the optimal panel design considerations when using CD21 Antibody, FITC in multiparameter flow cytometry?

When incorporating CD21 Antibody, FITC into multiparameter flow cytometry panels, researchers should consider several technical factors to ensure optimal data quality:

Spectral Considerations:

• FITC has excitation/emission maxima of approximately 495 nm/524 nm

• Avoid fluorochromes with substantial spectral overlap in the same panel (PE, FITC)

• If using multiple green fluorochromes, ensure proper compensation

Panel Design Strategy:

• Reserve FITC-CD21 for identifying B cell subsets or follicular dendritic cells

• Pair with markers that define developmental stages (IgD, IgM, CD27) for comprehensive B cell phenotyping

• For T cell studies focusing on the CD21+ subset, combine with CD3, CD4/CD8, and memory/activation markers

Technical Optimization:

• Titrate the antibody specifically in your experimental system (recommended starting point: 5 μl per 10^6 cells or 5 μl per 100 μl whole blood)

• For mouse samples using 7G6 clone, remember that fixation dramatically reduces signal intensity

• Include proper controls, especially when studying cells with variable or low CD21 expression

Sample-specific Considerations:

• For human PBMCs, CD21 expression is relatively robust on B cells

• Expression levels vary significantly across different immune contexts (inflammation, activation, disease states)

• Consider fixation alternatives when studying activated lymphocytes to preserve both viability and staining intensity

How can CD21 Antibody, FITC be used to study Epstein-Barr virus infection mechanisms?

CD21 serves as the primary receptor for Epstein-Barr virus (EBV), making CD21 Antibody, FITC an invaluable tool for investigating EBV infection mechanisms through several methodological approaches:

Receptor Expression Analysis:

• Quantify CD21 expression levels on potential target cells to correlate with EBV susceptibility

• Monitor changes in CD21 expression during various stages of EBV infection

• Compare CD21 density on EBV-susceptible versus resistant B cell populations

Receptor Blocking Studies:

• Pre-treat cells with unlabeled CD21 antibodies before EBV exposure to assess receptor dependency

• Use CD21 Antibody, FITC to confirm binding site competition with viral components

• Correlate degree of CD21 saturation with infection efficiency

Infection Kinetics:

• Monitor CD21 internalization patterns following EBV exposure using time-course flow cytometry

• Assess co-localization of CD21 with viral proteins during early infection events

• Investigate how CD21/CD19/CD81 complex dynamics change during EBV entry

Clinical Research Applications:

• Compare CD21 expression profiles between healthy donors and patients with EBV-associated disorders

• Correlate CD21 expression patterns with viral load and disease progression

• Study how genetic variants of CD21 affect susceptibility to EBV-related complications

For these applications, researchers should note that CD21 binds to the EBV glycoprotein gp350/220, facilitating viral attachment and subsequent fusion events. The antibody can help elucidate these molecular interactions when used in conjunction with viral tracking methods.

What methodological approaches can resolve contradictory data when studying CD21 expression across immune cell subsets?

When researchers encounter contradictory data regarding CD21 expression across immune cell subsets, several methodological approaches can help resolve discrepancies:

1. Standardized Sample Processing:

• Implement consistent isolation protocols to minimize artificial changes in expression

• Process all comparative samples simultaneously using identical reagents and conditions

• Document time delays between sample collection and analysis

2. Rigorous Antibody Validation:

• Compare multiple antibody clones targeting different CD21 epitopes

• Verify specificity using CD21-knockout controls or knockdown systems

• Perform epitope mapping to ensure detected signals correspond to genuine CD21

3. Comprehensive Controls:

• Include fluorescence minus one (FMO) controls to establish accurate gating

• Use isotype controls matched to the specific CD21 antibody's host/isotype (Mouse/IgG1, kappa for many CD21 antibodies)

• Include positive control samples with well-established CD21 expression patterns

4. Multi-platform Verification:

• Supplement flow cytometry data with protein detection methods (Western blot, immunoprecipitation)

• Confirm expression at the transcript level using RT-PCR or RNA sequencing

• Employ imaging techniques to visualize CD21 localization patterns

5. Biological Context Considerations:

• Document activation state of cells, as CD21 expression decreases upon B cell activation

• Note developmental stage, as CD21 appears after pre-B stage and is lost upon plasma cell differentiation

• Consider species differences in expression patterns between human and mouse systems

6. Detailed Experimental Reporting:

• Document antibody clone, fluorochrome, manufacturer, and lot number

• Report exact staining protocols, including buffer composition and incubation conditions

• Specify cytometer configuration, laser power, and detector voltages

By implementing these approaches, researchers can better understand whether contradictory data stems from technical variables, biological diversity, or contextual differences in CD21 expression regulation.

What are the optimal storage and handling conditions for CD21 Antibody, FITC?

For maximal stability and performance of CD21 Antibody, FITC, adhere to these storage and handling guidelines:

Storage Conditions:

• Store at 2-8°C (refrigerated, not frozen)

• Protect from light exposure at all times to prevent photobleaching of the FITC fluorochrome

• Maintain in the original container with proper sealing

• The antibody is typically stable for one year after shipment when stored correctly

Buffer Composition:

• Most CD21 Antibody, FITC preparations are supplied in PBS with 0.09% sodium azide and 0.5% BSA

• Sodium azide serves as a preservative but is toxic; dilute azide compounds in running water before disposal to prevent accumulation of potentially explosive deposits in plumbing

• The BSA component helps maintain antibody stability and reduce non-specific binding

Handling Precautions:

• Minimize freeze-thaw cycles, which can degrade antibody quality

• When aliquoting, prepare single-use volumes to avoid repeated thawing

• Centrifuge vials briefly before opening to collect liquid at the bottom

• Always use appropriate personal protective equipment when handling

Working Solution Preparation:

• Allow the antibody to equilibrate to room temperature before opening

• Mix gently by inversion or gentle pipetting, avoiding vigorous vortexing

• If dilution is required, use recommended buffers (typically PBS with 0.5-1% BSA)

• Use diluted solutions within the same day for optimal performance

How should researchers troubleshoot weak or absent CD21 staining in flow cytometry experiments?

When encountering weak or absent CD21 staining in flow cytometry experiments, implement the following troubleshooting methodology:

1. Sample Integrity Assessment:

• Verify cell viability using appropriate viability dyes

• Confirm proper sample handling to minimize time between collection and staining

• For mouse studies, remember CD21 expression is absent on thymocytes, T cells, erythrocytes, and platelets

2. Antibody-Specific Considerations:

• Check antibody expiration date and storage conditions

• Verify proper dilution (recommended: 5 μl per 10^6 cells in 100 μl or 5 μl per 100 μl whole blood)

• For FITC-conjugated 7G6 clone, remember staining intensity is severely reduced after fixation

3. Technical Protocol Evaluation:

• Ensure cytometer laser and detector settings are appropriate for FITC (488nm excitation)

• Verify compensation settings if using multiple fluorochromes

• Check for potential blocking antibodies or Fc receptors causing interference

• Ensure adequate incubation time and temperature

4. Biological Variables:

• Consider CD21 downregulation due to B cell activation status

• Note developmental stage, as CD21 appears after pre-B stage and disappears in plasma cells

• Assess pathological conditions that might alter CD21 expression

5. Procedural Modifications:

• Try alternative CD21 antibody clones or conjugates

• Implement signal amplification techniques if expression is low

• For mouse samples with 7G6 clone, analyze without fixation, keeping freshly-isolated cells in wash buffer at 4°C (stable up to 18 hours)

• For activated lymphocytes, collect data within 5 hours post-staining due to potential viability loss

6. Positive Control Implementation:

• Include samples known to express CD21 (e.g., human PBMCs with identified B cell populations)

• Consider using cell lines with stable CD21 expression as technical controls

• Run parallel staining with alternative B cell markers to confirm proper identification of target populations

How can CD21 Antibody, FITC be used to investigate B cell abnormalities in autoimmune disorders?

CD21 Antibody, FITC provides valuable methodological approaches for investigating B cell abnormalities in autoimmune disorders through several research applications:

1. Identification of CD21-low B Cell Populations:

• In several autoimmune conditions, expanded populations of CD21-low B cells have been identified

• Use CD21 Antibody, FITC in combination with other B cell markers to enumerate and phenotype these cells

• Apply standardized gating strategies to ensure consistent identification of CD21-low subsets

2. Correlation with Disease Parameters:

• Assess CD21 expression levels on B cells in relation to disease activity indices

• Track CD21-low B cell frequency longitudinally during disease progression or treatment

• Compare CD21 expression patterns between affected tissues and peripheral blood

3. Functional Characterization:

• Sort CD21-high versus CD21-low B cell populations using FITC-CD21 for downstream functional assays

• Investigate differences in activation threshold, cytokine production, and antibody secretion

• Assess BCR signaling strength differences between CD21-normal and CD21-low populations

4. Complement Interaction Studies:

• Examine how complement fragment binding to CD21 differs in autoimmune contexts

• Investigate whether CD21 expression changes correlate with altered complement activation

• Use competition assays with labeled complement fragments to assess receptor functionality

5. Treatment Response Monitoring:

• Evaluate how B cell-targeted therapies affect CD21 expression patterns

• Determine whether normalization of CD21 expression correlates with clinical improvement

• Identify potential CD21-related biomarkers predictive of treatment response

For optimal results, researchers should employ multiparameter approaches that combine CD21 Antibody, FITC with markers of B cell activation, differentiation, and function. This enables comprehensive characterization of B cell abnormalities within the complex immunological landscape of autoimmune disorders.