CD86 Antibody

What is CD86 and why is it important in immunological research?

CD86 (also known as B7-2) is a critical costimulatory molecule with an approximate mass of 70 kDa that plays a fundamental role in immune regulation. It functions as a receptor involved in providing the costimulatory signal essential for T-lymphocyte proliferation and interleukin-2 production by binding CD28 or CTLA-4 . CD86 is particularly important in early events of T-cell activation and costimulation of naive T-cells, helping determine between immunity and anergy within 24 hours after activation . Additionally, CD86 regulates B cell function and influences IgG1 production levels . The molecule is predominantly expressed on antigen-presenting cells, including activated B-lymphocytes, monocytes, and dendritic cells, making it a valuable marker for immunological studies .

What are the key considerations when selecting a CD86 antibody for flow cytometry?

When selecting a CD86 antibody for flow cytometry applications, researchers should consider several critical factors. First, verify the antibody has been validated specifically for flow cytometry applications, as seen with antibodies like clone BU63 (ab234000) which has demonstrated effectiveness in this application . Second, confirm the species reactivity aligns with your experimental model; many commercially available antibodies are specified for human or mouse samples . Third, consider the fluorophore conjugation that complements your existing panel design to avoid spectral overlap with other markers. Finally, evaluate published literature demonstrating the antibody's performance in similar experimental contexts to yours . When designing multicolor panels, CD86 antibodies can effectively identify activated dendritic cells and other antigen-presenting cells, as evidenced by researchers who have successfully used these antibodies to monitor activation of myeloid/APC cells during various experimental conditions including M. tuberculosis infection .

How do I validate CD86 antibody specificity for my experiments?

Validation of CD86 antibody specificity is critical for reliable experimental results. A comprehensive validation approach includes multiple methods. First, perform comparative analysis using knockout cell lines, such as B7-2/CD86 knockout Ramos human lymphoma cell lines, which should show no staining with CD86 antibodies while parental cell lines show positive staining . Second, conduct western blot analysis to confirm the antibody detects proteins of the expected molecular weight (approximately 70-75 kDa for CD86) . Third, utilize positive and negative control samples in your experimental system; for instance, unstimulated versus stimulated dendritic cells, where CD86 expression should increase following activation . Fourth, include proper isotype controls in flow cytometry experiments to establish background staining levels . Finally, comparative testing with alternative validated CD86 antibody clones can further confirm specificity. These approaches collectively provide robust validation of antibody specificity and ensure reliable experimental outcomes.

Which application methods are most effective for detecting CD86 in different sample types?

Different experimental approaches offer distinct advantages for CD86 detection depending on sample type and research question. For cell surface expression analysis, flow cytometry provides quantitative measurement of CD86 on individual cells and enables multi-parameter analysis with other markers, particularly effective for peripheral blood mononuclear cells, dendritic cells, and B lymphocytes . Western blot analysis effectively demonstrates CD86 protein expression in cell lysates, with studies detecting specific bands at approximately 75 kDa under reducing conditions, as demonstrated with Daudi and Ramos cell lines . Immunohistochemistry on formalin-fixed, paraffin-embedded tissues allows visualization of CD86 expression in tissue context, with successful applications in tumor samples at concentrations of approximately 4 μg/ml . For functional studies, neutralization assays can assess the role of CD86 in T-cell activation, measuring outcomes such as IL-2 secretion by Jurkat cells following CD86 engagement, with typical neutralization doses (ND₅₀) of 0.5-2.5 μg/mL . Each method provides complementary information, and selection should be based on specific experimental endpoints and sample characteristics.

How can I optimize CD86 antibody staining protocols for dendritic cell maturation studies?

Optimizing CD86 antibody staining for dendritic cell maturation studies requires attention to several methodological details. Begin with proper dendritic cell preparation; for bone marrow-derived dendritic cells (BMDCs), standard differentiation protocols using GM-CSF and IL-4 provide consistent populations . When analyzing maturation, include appropriate stimulation controls such as LPS (100 ng/mL), poly I:C, or CD40L to induce CD86 upregulation . For flow cytometry, use antibody concentrations of 1-5 μg/mL for optimal signal-to-noise ratio, and include additional maturation markers such as CD80, CD40, and MHC II to comprehensively assess activation status . Co-staining with dendritic cell identification markers (CD11c for mouse, CD1c for human) ensures proper gating strategy . Temperature and timing are critical; perform staining at 4°C for 30 minutes to prevent receptor internalization, and process samples consistently across experimental conditions. For kinetic studies of maturation, include multiple time points (6, 12, 24, 48 hours post-stimulation) to capture the dynamics of CD86 upregulation. Finally, quantitative analysis using mean fluorescence intensity (MFI) rather than percent positive cells provides more sensitive detection of expression level changes during the maturation process.

What are the best practices for using CD86 antibodies in immunohistochemistry (IHC)?

Successful immunohistochemical detection of CD86 requires careful optimization of several parameters. For formalin-fixed, paraffin-embedded (FFPE) tissues, antigen retrieval is critical; heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) for 20 minutes typically yields optimal results . The antibody concentration should be titrated for each tissue type and fixation method, with published protocols suggesting 4 μg/ml as an effective starting concentration for human tissues . When analyzing lymphoid tissues or tumors, inclusion of appropriate positive controls (such as tonsil sections containing activated B cells and dendritic cells) and negative controls (isotype-matched or primary antibody omission) is essential for validation. Detection systems may influence sensitivity; amplification methods such as polymer-based systems often provide superior signal compared to traditional ABC methods. For dual or multiplex staining to co-localize CD86 with other immune markers, sequential staining protocols with proper blocking steps between antibodies are recommended to prevent cross-reactivity. Finally, quantification methods should be standardized based on staining intensity and percentage of positive cells, with clear documentation of scoring criteria to ensure reproducibility across observers and experiments.

How can CD86 antibodies be utilized in single-cell analysis technologies beyond flow cytometry?

CD86 antibodies can be powerfully integrated into advanced single-cell technologies that extend beyond conventional flow cytometry. In mass cytometry (CyTOF), metal-conjugated anti-CD86 antibodies enable high-dimensional profiling of immune cell activation states in combination with 30+ additional markers, providing comprehensive phenotyping of antigen-presenting cells without fluorescence spillover concerns . For spatial analysis, imaging mass cytometry and multiplexed immunofluorescence techniques incorporating CD86 antibodies reveal the spatial relationships between CD86+ antigen-presenting cells and T cells within tissue microenvironments, offering insights into immunological synapse formation in situ . Single-cell RNA sequencing paired with protein expression analysis (CITE-seq) using oligo-tagged CD86 antibodies correlates surface CD86 protein levels with transcriptional profiles at single-cell resolution, revealing heterogeneity within seemingly uniform cell populations . Additionally, the diffusional mobility shift assay employs CD86 antibodies to analyze transient membrane protein interactions in live cells, as demonstrated in studies examining dynamic receptor-ligand binding kinetics . These advanced applications expand the utility of CD86 antibodies beyond simple detection, enabling integrated multi-parameter analysis of immune cell function, spatial organization, and molecular interactions at unprecedented resolution.

What strategies can address variable CD86 expression in different experimental models?

Addressing variable CD86 expression across experimental models requires strategic approaches tailored to specific research contexts. For in vitro studies with primary human cells, donor-to-donor variability can be substantial; implementing standardized isolation protocols and pooling cells from multiple donors can minimize this variation . When comparing CD86 expression between different cell types (e.g., B cells versus dendritic cells), normalization to cell type-specific housekeeping proteins rather than global housekeeping genes provides more accurate relative quantification in western blot analyses . For studies involving in vivo models, controlling for microenvironmental factors that influence CD86 expression is essential; this includes standardizing housing conditions for laboratory animals and documenting clinical parameters for patient-derived samples . The detection of soluble CD86 forms in plasma, which are elevated in certain leukemia patients, requires optimization of ELISA protocols with specific antibody pairs that distinguish between membrane-bound and soluble isoforms . Temporal dynamics further complicate CD86 analysis; implementing time-course studies with consistent sampling intervals captures expression kinetics following stimulation or during disease progression. Finally, when working with tissue samples, accounting for regional heterogeneity through systematic sampling and quantitative image analysis ensures representative assessment of CD86 expression across the entire specimen.

How do CD86 antibodies perform in neutralization assays for T cell activation studies?

CD86 neutralizing antibodies provide powerful tools for dissecting costimulatory pathways in T cell activation studies, but require careful experimental design for meaningful results. Functional neutralization assays typically use recombinant CD86 to induce IL-2 secretion in Jurkat cells or primary T cells, with neutralizing antibodies demonstrating dose-dependent inhibition . The neutralization dose (ND₅₀) for effective antibodies ranges from 0.5-2.5 μg/mL in the presence of 2 μg/mL recombinant human B7-2/CD86 Fc chimera . For optimal experimental design, include both positive controls (maximal T cell activation without neutralizing antibody) and negative controls (isotype-matched antibodies) to distinguish specific CD86 blockade from non-specific effects . When studying the relative contributions of CD86 versus CD80 in costimulation, comparative and combinatorial neutralization approaches reveal synergistic or redundant functions. It's critical to recognize context-dependent performance variations; neutralizing efficiency may differ between plate-bound and cell-expressed CD86, between different T cell subsets, and in the presence of additional costimulatory or coinhibitory signals. Finally, readouts beyond IL-2 secretion, including cell proliferation (CFSE dilution), expression of activation markers (CD25, CD69), and production of multiple cytokines, provide comprehensive assessment of T cell activation states following CD86 neutralization.

What are common causes of inconsistent CD86 antibody staining in flow cytometry?

Inconsistent CD86 antibody staining in flow cytometry can stem from multiple technical and biological factors. Sample preparation variables significantly impact results; erythrocyte lysis procedures may reduce monocyte CD86 expression, while mechanical dissociation of tissues can cleave surface CD86 . Fixation conditions are critical; overfixation with high paraformaldehyde concentrations (>2%) or extended fixation times can mask the CD86 epitope, while inadequate fixation may alter membrane integrity . Antibody-specific factors include lot-to-lot variability, requiring validation of each new lot against previous standards, and suboptimal antibody concentration, necessitating titration experiments to determine ideal staining conditions (typically 1-5 μg/mL) . Cell-intrinsic factors also contribute to variability; CD86 expression fluctuates with activation state and is rapidly modulated by cytokines and microbial products, making timing of analysis critical . Technical aspects of cytometer setup, including insufficient compensation for spectral overlap when using multiple fluorochromes, can generate false signals . Additionally, non-specific binding can be addressed through careful blocking procedures and appropriate isotype controls . Systematic troubleshooting through controlled modification of each variable helps identify and resolve specific causes of inconsistent staining.

How do I address background staining issues when using CD86 antibodies?

Background staining with CD86 antibodies can compromise data interpretation, but several strategies effectively minimize this issue. First, implement proper blocking protocols using species-appropriate serum (5-10%) or commercial blocking reagents for 15-30 minutes before antibody incubation to reduce non-specific binding . For Fc receptor-expressing cells like monocytes and B cells, include specific Fc receptor blocking agents (human: 10% human serum or commercial Fc block; mouse: anti-CD16/CD32 antibodies) to prevent Fc-mediated antibody binding . Optimize antibody concentration through titration experiments; excessive antibody concentrations increase background while insufficient amounts reduce specific signal, with optimal concentrations typically in the 1-5 μg/mL range for most applications . When performing multicolor flow cytometry, include fluorescence-minus-one (FMO) controls to establish proper gating thresholds that account for spectral spillover . For immunohistochemistry applications, incorporate additional blocking steps for endogenous peroxidase or alkaline phosphatase activity, and consider tissue-specific autofluorescence quenching methods for immunofluorescence . Finally, if persistent background occurs with one antibody clone, evaluating alternative clones can identify those with superior signal-to-noise characteristics for your specific application .

What factors affect CD86 protein detection in western blot applications?

Successful CD86 detection in western blot applications depends on optimizing multiple technical parameters. Sample preparation is critical; CD86 is heavily glycosylated, resulting in apparent molecular weights of approximately 70-75 kDa rather than its predicted 37.7 kDa core protein mass . Lysis buffer composition significantly impacts extraction efficiency; buffers containing 1% NP-40 or Triton X-100 with protease inhibitors are generally effective for membrane protein solubilization . For optimal detection, reducing conditions are recommended, as demonstrated in protocols using Immunoblot Buffer Group 1 . Protein loading amounts require careful optimization; 20-50 μg of total protein per lane is typically sufficient, but may need adjustment based on CD86 expression levels in specific cell types . Transfer conditions should be optimized for high-molecular-weight glycoproteins, potentially including extended transfer times or reduced methanol concentrations in transfer buffers . Antibody incubation parameters significantly influence results; primary antibody concentrations of approximately 5 μg/mL have proven effective, with overnight incubation at 4°C often yielding superior results to shorter incubations . When analyzing specific cell lines, reference data from previous studies can guide expectations; CD86 has been successfully detected in Daudi and Ramos human Burkitt's lymphoma cell lines, with knockout cell lines serving as valuable negative controls .

How are CD86 antibodies utilized in investigations of immunotherapy response?

CD86 antibodies provide valuable tools for investigating immunotherapy response mechanisms across multiple research contexts. In checkpoint inhibitor studies, CD86 antibodies help quantify expression on antigen-presenting cells before and after treatment, revealing potential correlations between CD86 levels and clinical outcomes . Flow cytometric analysis of peripheral blood mononuclear cells using CD86 antibodies in conjunction with other activation markers assesses systemic changes in antigen-presenting cell activation status following immunotherapy . In tumor microenvironment investigations, immunohistochemical staining with CD86 antibodies on sequential biopsies visualizes spatial and temporal changes in CD86-expressing cells within the tumor, particularly valuable for evaluating dendritic cell infiltration and activation . Mechanistic studies employ neutralizing CD86 antibodies in ex vivo functional assays to determine how blockade affects T cell activation in patient-derived samples, providing insights into costimulatory pathway contributions to treatment response . Additionally, detecting soluble CD86 forms in patient plasma using specialized ELISA assays may provide biomarkers of immune activation, with studies showing elevated levels in some leukemia patients . These diverse applications of CD86 antibodies collectively enhance understanding of immunotherapy response mechanisms and may identify predictive biomarkers for patient stratification.

What considerations are important when analyzing CD86 expression in clinical tumor samples?

Analysis of CD86 expression in clinical tumor samples requires careful attention to multiple pre-analytical, analytical, and interpretative factors. Pre-analytical variables significantly impact results; standardized tissue collection, fixation times (typically 24-48 hours in 10% neutral buffered formalin), and processing protocols are essential for consistent immunohistochemical CD86 detection . Antigen retrieval methods must be optimized for each tumor type; HIER using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) for 20 minutes provides effective epitope recovery in most formalin-fixed, paraffin-embedded samples . Antibody validation using positive controls (lymphoid tissues containing activated B cells and dendritic cells) and negative controls (CD86-negative tissues or isotype controls) establishes staining specificity . Quantification approaches should address tumor heterogeneity through systematic sampling of multiple regions, with scoring systems that account for both staining intensity and percentage of positive cells . Interpretation requires cellular context; distinguishing between CD86 expression on tumor cells versus infiltrating immune cells necessitates dual staining with lineage-specific markers . Clinical correlation demands consistent timing of sample collection relative to treatment, as therapy may alter CD86 expression patterns . Finally, comparative analysis across patient cohorts requires standardized protocols and scoring systems to minimize inter-laboratory and inter-observer variability, essential for developing CD86 as a potential prognostic or predictive biomarker.

How do CD86 antibodies contribute to understanding autoimmune disease mechanisms?

CD86 antibodies provide critical insights into autoimmune disease pathophysiology through multiple research applications. Flow cytometric analysis with CD86 antibodies reveals altered expression patterns on antigen-presenting cells in various autoimmune conditions, with studies demonstrating heightened CD86 expression on dendritic cells and B cells in systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis . Immunohistochemical investigations using CD86 antibodies visualize the distribution and activation state of costimulatory molecule-expressing cells within affected tissues, such as synovium in arthritis or central nervous system lesions in multiple sclerosis . Functional studies employ neutralizing CD86 antibodies in ex vivo systems to assess the contribution of this costimulatory pathway to autoreactive T cell activation, providing mechanistic insights and potential therapeutic targets . Detection of soluble CD86 forms in patient plasma using specialized ELISA methods offers potential biomarkers of disease activity, with research indicating altered levels in certain autoimmune conditions . The utility of CD86 antibodies extends to therapeutic monitoring; flow cytometric analyses track changes in CD86 expression following immunomodulatory treatments, potentially identifying cellular mechanisms of treatment response or resistance . Additionally, CD86 antibodies facilitate investigation of how environmental factors influence costimulatory molecule expression, contributing to understanding of gene-environment interactions in autoimmune disease pathogenesis.