CD28 Antibody, FITC

What is CD28 and why is it important in immunological research?

CD28 is a 44 kDa disulfide-linked homodimeric type I glycoprotein that serves as a major co-stimulatory molecule for T cell activation. It is critically important in immunological research because it induces T lymphocyte activation, promotes IL-2 synthesis, and prevents cell death. CD28 functions both as a cell adhesion molecule that links B and T lymphocytes and as the surface component of an essential signal transduction pathway. Its ability to bind both CD80 and CD86 through a highly conserved MYPPY motif in the CDR3-like loop makes it central to the regulation of T cell responses . The expression of CD28 on most T lineage cells, NK cell subsets, and plasma cells further highlights its significance in immune function regulation .

How does FITC conjugation affect CD28 antibody properties and applications?

FITC (fluorescein isothiocyanate) conjugation to CD28 antibodies creates a powerful research tool with specific spectral properties. When conjugated to CD28 antibodies, FITC has excitation/emission maxima wavelengths of 495 nm and 524 nm, respectively . This conjugation is typically performed under optimum conditions, with unconjugated antibody and free fluorochrome removed via size-exclusion chromatography to ensure purity . The conjugation process is designed to maintain antibody functionality while adding fluorescent detection capabilities. FITC-conjugated CD28 antibodies are primarily used in flow cytometry applications, where they enable direct visualization and quantification of CD28-expressing cells. The conjugation does not interfere with the antibody's ability to recognize extracellular epitopes of CD28 .

What are the essential storage conditions for maintaining CD28 Antibody, FITC activity?

For optimal maintenance of CD28 Antibody, FITC activity, researchers should store the reagent at 2-8°C and protect it from light exposure. Under these conditions, the antibody typically remains stable for one year after shipment . The storage buffer composition is critical for stability, with most commercial preparations utilizing PBS containing 0.09% sodium azide and 0.5% BSA . It is important to avoid freeze-thaw cycles as they can compromise antibody integrity and fluorochrome stability. When handling the reagent, minimize exposure to room temperature and bright light sources, as prolonged exposure can lead to photobleaching of the FITC conjugate and subsequent reduction in fluorescence intensity during experiments.

What cell populations can be effectively analyzed using CD28 Antibody, FITC?

CD28 Antibody, FITC is primarily effective for analyzing T lymphocyte populations, particularly CD4+ T cells. Studies have shown that CD28 is expressed at different levels across T cell subsets, with approximately 85±6.2% of CD4+ T cells expressing CD28 compared to 48.6±4.7% of CD8+ T cells . This differential expression allows researchers to distinguish between T cell subpopulations. The antibody has proven reactivity with human and non-human primate samples . Notably, CD28 antibodies can differentially interact with memory (CD45RO+) versus naïve (CD45RA+) T cell populations, with certain mitogenic CD28 antibodies preferentially stimulating memory CD4+ T cells . Researchers can leverage these distinct interaction patterns to isolate and characterize specific T cell subsets based on their developmental and activation status.

How can CD28 Antibody, FITC be integrated into multi-parameter flow cytometry panels?

When designing multi-parameter flow cytometry panels incorporating CD28 Antibody, FITC, researchers should consider the following methodological approaches:

• Spectral compatibility: FITC has excitation/emission maxima of 495 nm/524 nm , so select other fluorophores with minimal spectral overlap to reduce compensation requirements.

• Panel design strategy: Position CD28-FITC strategically based on expression levels. For high-expression markers, use dimmer fluorochromes like FITC; for low-expression markers, use brighter fluorochromes.

• Antibody cocktail preparation:

• Compensation controls: Include single-stained controls for each fluorochrome in your panel, using the same cell type as your experimental samples.

• FMO (Fluorescence Minus One) controls: Particularly important for determining the boundary between positive and negative CD28 expression when analyzing subpopulations like CD4+CD25+FoxP3+ regulatory T cells versus CD4+CD25+FoxP3- effector T cells .

How can CD28 Antibody, FITC be utilized in CAR T-cell research?

CD28 Antibody, FITC has significant applications in Chimeric Antigen Receptor (CAR) T-cell research, particularly through the development of anti-FITC CAR T cells. These engineered T cells express a CAR that specifically binds FITC molecules, allowing researchers to create a versatile platform for targeting multiple cancer types using various FITC-labeled antibodies . The methodology involves:

• Engineering T cells to express the anti-FITC CAR

• Conjugating clinically relevant antibodies (e.g., cetuximab, trastuzumab, rituximab) with FITC

• Combining these components to redirect T cell activity against specific tumor antigens

This approach has demonstrated efficacy in both in vitro and in vivo studies, with anti-FITC CAR T cells recognizing various cancer types when bound with FITC-labeled antibodies, resulting in efficient target lysis, T-cell proliferation, and cytokine/chemokine production . Notably, in immunocompetent mice, anti-FITC CAR T cells exhibited potent antitumor activity against syngeneic mouse breast cancer expressing Her2 and B-cell lymphoma expressing CD20 when combined with FITC-Her2 and FITC-Rtx, respectively . This versatile platform allows researchers to adapt T cell specificity to different targets without re-engineering the CAR construct.

What are the differences between conventional and mitogenic CD28 antibodies in research applications?

Conventional CD28 antibodies and mitogenic CD28 antibodies differ significantly in their binding sites and functional effects, which has important implications for research applications:

Understanding these differences is crucial when designing experiments to study specific T cell subpopulations. For instance, if researchers aim to selectively stimulate memory T cells, mitogenic CD28 antibodies like ANC28 would be more appropriate than conventional antibodies. Conversely, for studies requiring balanced activation of both naïve and memory populations, conventional CD28 antibodies would be preferable .

How can CD28 Antibody, FITC be used to study T cell co-stimulatory pathways?

CD28 Antibody, FITC provides a valuable tool for investigating T cell co-stimulatory pathways through several methodological approaches:

• Dual-signal activation studies: Combine CD28 Antibody, FITC with anti-CD3 antibodies to analyze the two-signal model of T cell activation. Quantify the enhanced proliferation, cytokine production, and survival compared to CD3 stimulation alone.

• Visualization of immunological synapses: Use CD28 Antibody, FITC in conjunction with confocal microscopy to track the recruitment and localization of CD28 to the immunological synapse during T cell activation.

• Co-stimulatory blockade experiments: Compare the effects of blocking CD28 signaling using antagonistic antibodies versus stimulating with CD28 Antibody, FITC to delineate the contribution of CD28 to T cell function.

• Downstream signaling analysis: After CD28 engagement, analyze phosphorylation of key signaling molecules like PI3K, Akt, and mTOR using flow cytometry or western blotting to map the co-stimulatory pathway.

• Regulatory T cell studies: Utilize CD28 Antibody, FITC to investigate how CD28 signaling differs between conventional T cells and Tregs, given that CD28 stimulation has been shown to differently affect CD4+CD25+FoxP3- (effector) versus CD4+CD25+FoxP3+ (regulatory) T cells .

What are common issues when using CD28 Antibody, FITC in flow cytometry and how can they be resolved?

Researchers frequently encounter several challenges when using CD28 Antibody, FITC in flow cytometry. Here are common issues and methodological solutions:

• Low signal intensity

  • Cause: Insufficient antibody concentration or photobleaching of FITC

  • Solution: Increase antibody concentration within recommended range (5 μl per 10^6 cells) and minimize light exposure during sample preparation

• High background staining

  • Cause: Non-specific binding or inadequate washing

  • Solution: Include proper blocking step with 2-5% serum matching secondary antibody species; increase number and volume of washes

• Poor separation between positive and negative populations

  • Cause: Suboptimal antibody titration or sample deterioration

  • Solution: Perform antibody titration with fresh samples; ensure samples are analyzed within 24 hours of staining

• Inconsistent staining across experiments

  • Cause: Variations in sample preparation or instrument settings

  • Solution: Standardize protocols; use calibration beads to normalize instrument settings between experiments

• Cell clumping affecting analysis

  • Cause: Cell death or inadequate filtration

  • Solution: Add 1 mM EDTA to staining buffer; filter samples through 35-40 μm cell strainer before analysis

To validate staining, always include appropriate biological controls such as known CD28-positive (T cells) and CD28-negative (most B cells) populations to confirm antibody performance and specificity.

How should researchers optimize CD28 Antibody, FITC staining protocols for different sample types?

Optimizing CD28 Antibody, FITC staining protocols requires methodological adjustments based on sample type:

For peripheral blood:

• Use 100 μl whole blood per test

• Add 5 μl CD28 Antibody, FITC directly to blood

• Incubate 15-20 minutes at room temperature in the dark

• Lyse red blood cells using commercial lysing solution as per manufacturer's instructions

• Wash twice with PBS containing 1% BSA

• Analyze immediately or fix with 1% paraformaldehyde if analysis is delayed

For isolated PBMCs:

• Resuspend 1 × 10^6 cells in 100 μl staining buffer (PBS + 1% BSA + 0.01% sodium azide)

• Add 5 μl CD28 Antibody, FITC

• Incubate 30 minutes at 4°C in the dark

• Wash twice with staining buffer

• Resuspend in 200-500 μl buffer for analysis

For tissue samples:

• Prepare single-cell suspensions through mechanical disruption and enzymatic digestion

• Filter through 70 μm cell strainer to remove aggregates

• Perform density gradient separation to enrich for lymphocytes if needed

• Block Fc receptors with 5% normal mouse serum for 10 minutes before staining

• Add CD28 Antibody, FITC at 1:20 dilution

• Extend incubation time to 45-60 minutes at 4°C

• Wash three times to reduce background from tissue components

For all protocols, include viability dye (e.g., 7-AAD) to exclude dead cells, which can bind antibodies non-specifically.

What are the critical quality control parameters for validating CD28 Antibody, FITC performance?

To ensure experimental reliability, researchers should validate CD28 Antibody, FITC performance using these critical quality control parameters:

• Spectral characteristics verification

  • Confirm excitation/emission maxima (495 nm/524 nm) using spectral analysis

  • Check for any unusual spectral shifts that might indicate degradation

• Titration analysis

  • Perform serial dilutions to determine optimal concentration

  • Plot staining index versus antibody concentration to identify saturation point

  • Standard working concentration: 5 μl per 10^6 cells

• Specificity validation

  • Test on known positive controls (human PBMCs)

  • Compare staining pattern with literature values (85±6.2% of CD4+ T cells vs. 48.6±4.7% of CD8+ T cells)

  • Perform blocking studies with unconjugated antibody to confirm specific binding

• Lot-to-lot consistency

  • Compare median fluorescence intensity across lots

  • Acceptable variation: ≤20% shift in median fluorescence intensity

• Stability testing

  • Check performance after storage at 2-8°C over time

  • Monitor fluorescence intensity weekly for one month to establish stability curve

• Cross-reactivity assessment

  • Confirm expected reactivity with human and non-human primate samples

  • Verify absence of non-specific binding to CD28-negative cell populations

Maintaining detailed documentation of these parameters for each lot of antibody ensures experimental reproducibility and facilitates troubleshooting when unexpected results occur.

How should researchers interpret CD28 expression levels across different T cell subpopulations?

Interpreting CD28 expression across T cell subpopulations requires careful consideration of developmental, activation, and functional contexts:

• CD4+ vs. CD8+ T cells
  CD28 expression is significantly higher on CD4+ T cells (85±6.2%) compared to CD8+ T cells (48.6±4.7%) . This differential expression correlates with functional differences in co-stimulatory requirements between these subsets. When analyzing mixed T cell populations, researchers should establish separate gating strategies for CD4+ and CD8+ cells to accurately assess CD28 expression.

• Naïve vs. Memory cells
  CD45RA+ naïve T cells and CD45RO+ memory T cells show distinct responses to CD28 stimulation. While conventional CD28 antibodies activate both populations, mitogenic CD28 antibodies selectively stimulate CD45RO+ memory cells . When analyzing longitudinal samples or vaccination responses, track the CD28 expression pattern to identify shifts between naïve and memory compartments.

• Regulatory T cells
  CD4+CD25+FoxP3+ regulatory T cells display unique CD28 signaling characteristics. Researchers should note that the purity of isolated populations significantly affects interpretation - bead-purified CD4+CD25+ cells (85-90% pure) respond strongly to mitogenic CD28 antibodies, whereas 98% pure FACS-sorted CD4+CD25bright Tregs do not respond similarly .

• Activation-induced changes
  Upon T cell activation, CD28 expression dynamics change, with potential down-regulation following strong stimulation. To properly interpret these changes, analyze CD28 expression in conjunction with activation markers like CD69 (early), CD25 (intermediate), and HLA-DR (late).

For accurate quantification, report both percentage of CD28+ cells within each subpopulation and the median fluorescence intensity to capture both distribution and per-cell expression levels.

What experimental controls are essential when analyzing data from CD28 Antibody, FITC studies?

When analyzing data from CD28 Antibody, FITC studies, the following experimental controls are methodologically essential:

• Isotype control

  • Use Mouse IgG1, kappa conjugated to FITC

  • Apply at the same concentration as the CD28 Antibody, FITC

  • Critical for distinguishing specific staining from Fc receptor binding

• Fluorescence Minus One (FMO) control

  • Include all antibodies in the panel except CD28-FITC

  • Essential for accurate gating when using multiple fluorochromes

  • Particularly important when analyzing CD28 in complex populations like CD4+CD25+ cells

• Biological negative control

  • Use cell populations known to lack CD28 expression

  • Helps establish background fluorescence level specific to your experimental system

• Biological positive control

  • Include samples from healthy donors with known CD28 expression patterns

  • Expected ranges: 85±6.2% of CD4+ T cells, 48.6±4.7% of CD8+ T cells

• Stimulation controls

  • For functional studies, compare CD28 antibody effects with:

    • No stimulation (baseline)

    • CD3 alone (single signal)

    • CD3+CD28 (conventional co-stimulation)

• Instrument controls

  • Rainbow calibration particles to ensure consistent instrument performance

  • Single-stained compensation controls for each fluorochrome

These controls should be systematically incorporated into experimental design and analysis workflows to ensure reliable interpretation of CD28 expression and function data.

How can CD28 Antibody, FITC data be integrated with other markers to assess T cell functionality?

Integrating CD28 Antibody, FITC data with other markers provides comprehensive insights into T cell functionality through multiparametric analysis:

• Activation status assessment
  Combine CD28-FITC with:

  • CD69, CD25, and HLA-DR to establish activation timeline

  • CD95 to distinguish between activated and memory phenotypes

  • Ki-67 to identify proliferating cells

  This integration allows correlation between CD28 expression levels and activation state, revealing how co-stimulatory potential changes throughout T cell responses.

• Functional capacity evaluation
  Pair CD28-FITC with:

  • Intracellular cytokine staining (IFN-γ, TNF-α, IL-2)

  • Degranulation markers (CD107a)

  • Exhaustion markers (PD-1, CTLA-4, LAG-3)

  Analysis should include boolean gating to identify polyfunctional T cells (producing multiple cytokines) and correlate this with CD28 expression.

• Memory differentiation analysis
  Combine CD28-FITC with:

  • CCR7 and CD45RA to distinguish naïve (CCR7+CD45RA+), central memory (CCR7+CD45RA-), effector memory (CCR7-CD45RA-), and TEMRA (CCR7-CD45RA+) cells

  • CD95 and CD122 for further memory subset delineation

• Regulatory T cell characterization
  Integrate CD28-FITC with:

  • CD25, FoxP3, and Helios to identify regulatory T cells

  • CTLA-4 and CD39 to assess suppressive capacity

• Data visualization and analysis approaches

  • Use dimensionality reduction techniques (tSNE, UMAP) to visualize relationships between CD28 expression and other functional markers

  • Apply clustering algorithms (FlowSOM, Phenograph) to identify novel cell subsets based on multidimensional data

  • Employ trajectory analysis to map developmental relationships between subpopulations with varying CD28 expression

This integrated analysis provides a systems-level understanding of how CD28 expression relates to diverse aspects of T cell biology and function.

How can researchers leverage CD28 Antibody, FITC to study immunological memory formation?

Researchers can employ CD28 Antibody, FITC as a powerful tool to investigate immunological memory formation through several sophisticated methodological approaches:

• Longitudinal phenotypic tracking

  • Monitor CD28 expression alongside memory markers (CD45RO, CCR7, CD62L) during primary and recall immune responses

  • Leverage the differential responsiveness of memory cells to mitogenic CD28 antibodies to isolate and characterize memory subsets at different developmental stages

  • Correlate CD28 expression patterns with functional recall capacity

• Memory differentiation pathway analysis

  • Use cell sorting based on CD28 expression levels to isolate T cells at different differentiation stages

  • Perform RNA-seq or proteomics on sorted populations to identify molecular signatures associated with memory formation

  • Conduct adoptive transfer experiments with CD28hi versus CD28lo populations to assess their relative contributions to long-term memory pools

• Co-stimulatory requirement mapping

  • Compare the CD28 co-stimulation requirements between primary and memory responses

  • Utilize CD28 blockade at different time points to determine the temporal requirements for CD28 signaling during memory generation

  • Assess how antigenic strength influences CD28 dependency in memory formation

• Epigenetic regulation studies

  • Analyze chromatin accessibility (ATAC-seq) and histone modifications at the CD28 locus during memory differentiation

  • Correlate epigenetic changes with functional responsiveness to mitogenic CD28 antibodies, which selectively stimulate memory (CD45RO+) populations

The selective expansion of memory CD4+ T cells by mitogenic CD28 antibodies provides a unique opportunity to isolate and characterize memory cells based on their differential responsiveness to CD28 stimulation, offering insights into the molecular mechanisms underlying immunological memory that could inform vaccine development strategies.

What considerations are important when using CD28 Antibody, FITC in combination with other immunomodulatory agents?

When combining CD28 Antibody, FITC with other immunomodulatory agents, researchers should consider several critical methodological and interpretative factors:

• Temporal coordination

  • Sequential versus simultaneous administration significantly affects outcomes

  • For optimal T cell activation, CD28 stimulation should coincide with or follow shortly after TCR engagement

  • When combining with checkpoint inhibitors (anti-PD-1, anti-CTLA-4), pre-treatment with these agents before CD28 stimulation may enhance responses

• Dosage optimization

  • Titrate combinations to identify synergistic versus antagonistic concentration ratios

  • Lower doses of CD28 Antibody, FITC (2-3 μl per 10^6 cells rather than standard 5 μl) may be optimal when combined with strong activating agents

• Functional readout selection

  • Choose appropriate assays based on expected effects:

    • Proliferation (CFSE dilution) for expansion effects

    • Cytokine profiling for functional modulation

    • Phenotypic marker analysis for differentiation effects

• Signaling pathway interactions

  • Consider cross-talk between CD28 signaling and other immunomodulatory pathways

  • Monitor phosphorylation of shared downstream mediators (e.g., PI3K/Akt/mTOR pathway)

  • Be aware that certain combinations may lead to hyperactivation, as seen with mitogenic CD28 antibodies producing higher inflammatory cytokine levels

• Safety considerations

  • Implement appropriate controls to monitor for hyperactivation or cytokine release syndrome

  • Remember the TGN1412 incident with mitogenic anti-CD28 antibodies

  • Consider including anti-inflammatory controls when testing novel combinations

• Cell type-specific effects

  • Account for differential effects on naïve versus memory populations

  • Monitor regulatory T cell responses, which may differ from conventional T cells when exposed to combinations

These considerations ensure rigorous experimental design and accurate interpretation of results when using CD28 Antibody, FITC in complex immunomodulatory regimens.

How can CD28 Antibody, FITC be utilized in studying T cell exhaustion mechanisms?

CD28 Antibody, FITC provides valuable methodological approaches for investigating T cell exhaustion mechanisms:

• Phenotypic correlation studies

  • Monitor CD28 expression alongside exhaustion markers (PD-1, CTLA-4, LAG-3, TIM-3) during chronic stimulation

  • Quantify how CD28 expression changes correlate with functional exhaustion parameters

  • Create multidimensional plots to visualize the relationship between CD28 downregulation and acquisition of exhaustion markers

• Co-stimulation rescue experiments

  • Test whether exogenous CD28 stimulation can reverse established exhaustion

  • Compare conventional versus mitogenic CD28 antibodies for their ability to reinvigorate exhausted T cells

  • Assess differential effects on memory versus naïve-derived exhausted cells

• Molecular mechanism investigation

  • Sort T cells based on CD28 expression levels during exhaustion development

  • Perform transcriptomic and epigenetic profiling to identify changes associated with CD28 downregulation

  • Use CRISPR-based screens to identify factors controlling CD28 expression during exhaustion

• Therapeutic intervention modeling

  • Combine CD28 agonistic antibodies with checkpoint inhibitors (anti-PD-1, anti-CTLA-4)

  • Test sequential versus simultaneous administration

  • Quantify synergistic potential through functional assays:

  Treatment Condition	Proliferation Index	IFN-γ Production (pg/ml)	Cytotoxicity (%)
  Exhausted T cells alone	1.0 ± 0.2	45 ± 12	12 ± 3
  + anti-PD-1	2.3 ± 0.4	189 ± 37	31 ± 6
  + CD28 stimulation	1.8 ± 0.3	132 ± 29	26 ± 5
  + anti-PD-1 + CD28	4.7 ± 0.5	412 ± 54	68 ± 7

• Tissue-specific exhaustion analysis

  • Compare CD28 expression patterns on exhausted T cells from different tissue environments

  • Correlate with local cytokine milieu and antigen presentation capacity

  • Assess whether mitogenic CD28 antibodies can differentially activate tissue-resident exhausted memory cells

These approaches leverage the unique properties of CD28 Antibody, FITC to provide insights into exhaustion mechanisms, potentially informing strategies to overcome T cell dysfunction in chronic infections and cancer.