MPP3 Antibody

What is MPP3 and why is it significant in hematopoietic research?

MPP3 refers to myeloid-biased multipotent progenitors, which represent a critical population in the hematopoietic stem cell hierarchy. Recent research has revealed that MPP3 cells are functionally and molecularly heterogeneous, containing a distinct subset of myeloid-primed secretory cells characterized by high endoplasmic reticulum (ER) volume and FcγR expression . These cells serve as a reservoir for rapid production of granulocyte/macrophage progenitors (GMPs) and directly amplify myelopoiesis through inflammation-triggered secretion of cytokines in the bone marrow microenvironment . The significance of MPP3 lies in its newly discovered regulatory function as a self-reinforcing amplification compartment in inflammatory stress and disease conditions, controlling myeloid differentiation through lineage-priming and cytokine production .

What are the key applications for MPP3 antibodies in research?

MPP3 antibodies are valuable tools for multiple research applications, including:

• Western Blotting (WB): For protein expression analysis and quantification of MPP3 in cell lysates

• Immunofluorescence (IF): To visualize the cellular localization of MPP3, particularly important for distinguishing ER high and ER low MPP3 subpopulations

• Flow Cytometry (FACS): Critical for isolating and characterizing MPP3 populations based on surface markers (e.g., FcγR expression)

• Immunohistochemistry (IHC): For tissue section analysis to study MPP3 distribution in different microenvironments

• Immunoprecipitation (IP): To study protein-protein interactions involving MPP3

These techniques allow researchers to investigate the multiple roles of MPP3 in hematopoiesis, cellular differentiation, and inflammatory responses.

How do I select the appropriate MPP3 antibody for my experiment?

Selection of an appropriate MPP3 antibody depends on several critical factors:

• Target epitope: Determine whether you need an N-terminal or C-terminal targeting antibody based on your research question. N-terminal antibodies (e.g., ABIN2786645) recognize the amino-terminal region of MPP3, which may be particularly useful for detecting full-length protein .

• Species reactivity: Verify the antibody's cross-reactivity with your species of interest. Some MPP3 antibodies show broad cross-reactivity across human, mouse, rat, and other species, with predictable reactivity percentages (e.g., 100% for human, mouse, rat; 93% for guinea pig) .

• Application compatibility: Confirm the antibody has been validated for your specific application (WB, IF, FACS, etc.) .

• Clonality: Consider whether a polyclonal antibody (offering multiple epitope recognition) or monoclonal antibody (offering high specificity) better suits your experimental needs .

• Validation data: Review available validation data, including positive controls used (e.g., cell lysates for Western blot) .

For studies focusing on secretory MPP3 populations, antibodies capable of distinguishing FcγR+/ER high MPP3 from FcγR-/ER low MPP3 are particularly valuable .

What markers are used alongside MPP3 antibodies to identify specific hematopoietic populations?

To properly identify and isolate MPP3 populations, researchers typically use a panel of markers in combination with MPP3 antibodies:

For specifically identifying the secretory MPP3 subset, additional markers are required:

• FcγR expression: Distinguishes myeloid-primed MPP3 subset

• ER-Tracker dye or KDEL (ER marker): Identifies ER high MPP3 secretory population

Approximately 31.0 ± 10.4% of the MPP3 compartment is identified as ER high MPP3 at steady state .

How can I distinguish between different MPP3 subpopulations in my research?

Distinguishing between different MPP3 subpopulations requires a multifaceted approach:

• Flow cytometry panel design: Combine Lin−/c-Kit+/Sca-1+/Flk2−/CD48+/CD150− markers with FcγR and ER-Tracker dye to separate:

  • FcγR+/ER high MPP3 (myeloid-primed secretory subset)

  • FcγR−/ER low MPP3 (immature subset)

• Morphological analysis: Transmission electron microscopy (TEM) can reveal distinct ER structures, with ER high MPP3 showing dense rough ER structures morphologically distinct from GMPs but similar to the secretory apparatus found in specialized immunoglobulin-producing plasma cells .

• Immunofluorescence microscopy: Using ER marker KDEL alongside MPP3 antibodies can provide visual confirmation of the ER high subset .

• Single-cell RNA sequencing: This approach reveals the molecular heterogeneity of MPP3, allowing identification of distinct clusters representing:

  • HSC gene-enriched immature group

  • GMP gene-enriched myeloid-primed group

  • Metabolically activated intermediate group

• Functional analysis: Secretory assays measuring cytokine production after LPS/Pam3CSK4 stimulation can functionally distinguish MPP3 subsets .

By combining these approaches, researchers can reliably identify and isolate the specific MPP3 subpopulation relevant to their research question.

What are the methodological considerations for studying secretory functions of MPP3?

To effectively study the secretory functions of MPP3, consider these methodological approaches:

• Stimulation protocols: Standard protocols use LPS/Pam3CSK4 for inflammatory stimulation before collecting supernatants at 24 hours .

• Secretome analysis options:

  • ELISA: For specific cytokines (e.g., TNFα, IL-6)

  • Cytokine arrays: For broad profiling (e.g., Raybiotech 200 mouse cytokine array)

  • Nanofluidic technology: For single-cell secretion analysis of preselected cytokines

• Key readouts to measure:

  • Pro-inflammatory cytokines: IL-1α, G-CSF, GM-CSF

  • Regulatory factors: TACI, CD40L

  • Autocrine regulators: IL-10 (suppresses IL-6 and TNFα)

• Controls: Include HSCs, MPP4, and GMPs as comparative populations to establish MPP3-specific secretory patterns .

• Downstream effects assessment: Perform differentiation assays with naïve HSPCs in methylcellulose and liquid culture using supernatants from stimulated versus unstimulated MPP3 .

Note that MPP3 exhibit complex cell type-specific secretory patterns with unique cytokine signatures dependent on stimulation status. Stimulated MPP3 show increased secretion of pro-inflammatory/pro-myeloid differentiation cytokines, while simultaneously modulating regulatory factors .

How can I troubleshoot inconsistent results when using MPP3 antibodies?

When encountering inconsistent results with MPP3 antibodies, implement this systematic troubleshooting approach:

• Antibody validation:

  • Confirm antibody specificity using positive controls (e.g., cell lysates expressing MPP3)

  • Perform peptide competition assays to verify epitope-specific binding

  • Consider testing multiple antibodies targeting different epitopes of MPP3

• MPP3 heterogeneity considerations:

  • Verify your isolation strategy correctly identifies your MPP3 population of interest

  • Remember that MPP3 comprises distinct subsets (FcγR+/ER high vs. FcγR−/ER low) with different molecular signatures

  • Confirm your gating strategy if using flow cytometry for isolation

• Experimental conditions:

  • MPP3 response to inflammatory stimuli is dynamic and time-dependent

  • Single-cell analyses reveal heterogeneous responses within the MPP3 population

  • Some cytokines (e.g., IL-10) can suppress the production of others (e.g., IL-6, TNFα) in a dose-dependent manner

• Technical optimization:

  • For Western blotting: Optimize protein extraction methods, blocking conditions, and antibody concentrations

  • For flow cytometry: Ensure proper compensation and gating strategies

  • For immunofluorescence: Optimize fixation and permeabilization protocols

• Biological variables:

  • MPP3 populations expand during myeloid regeneration (e.g., after anti-Ly6G antibody injection)

  • Different inflammatory conditions can significantly alter MPP3 behavior and marker expression

What methodologies are most effective for studying MPP3 in inflammatory conditions?

For studying MPP3 in inflammatory conditions, these methodologies have proven particularly effective:

• In vivo inflammatory models:

  • Granulocyte depletion model: Anti-Ly6G antibody injection drives myeloid regeneration, during which MPP3 expansion can be studied

  • This model specifically results in increased FcγR+ MPP3, confirming their role as an amplification compartment for myelopoiesis

• Ex vivo stimulation approaches:

  • Direct stimulation of isolated MPP3 with LPS/Pam3CSK4

  • Collection of supernatants after 24 hours for downstream analyses

• Analytical techniques:

  • Flow cytometry for quantifying shifts in MPP3 subpopulations

  • Secretome analysis for measuring cytokine production

  • Single-cell RNA sequencing for capturing transcriptional changes in response to inflammation

• Functional assays:

  • Differentiation assays using supernatants from stimulated versus unstimulated MPP3

  • Testing effects on naïve HSPCs in methylcellulose and liquid culture

• Time-course experiments:

  • Monitoring transient MPP3 expansion prior to GMP and myeloid cell expansion

  • Tracking the shift from ER low to ER high/FcγR+ MPP3 during inflammatory responses

These approaches allow for comprehensive analysis of how inflammatory signals alter MPP3 function, secretory activity, and contribution to emergency myelopoiesis.

How do I design experiments to investigate the molecular mechanisms underlying MPP3 function?

Designing robust experiments to investigate molecular mechanisms of MPP3 function requires a multifaceted approach:

• Transcriptional profiling strategies:

  • Bulk RNA sequencing to compare ER high versus ER low MPP3 populations

  • Single-cell RNA sequencing to identify distinct molecular clusters

  • K-means clustering analyses using highly variable genes (HVGs)

  • Principal component analyses to visualize relationships between MPP3 subsets and other hematopoietic populations (HSCs, GMPs)

• Functional genomics approaches:

  • Analysis of differentially expressed genes (DEGs) between MPP3 subsets

  • Focus on genes like Fcgr3 (myeloid-primed group) versus Gata2 and Meis1 (immature group)

  • Gene ontology (GO) analysis to identify enriched pathways

  • Slingshot analysis to predict differentiation trajectories

• Protein-level analysis:

  • Western blotting for key factors identified in transcriptional analyses

  • Immunoprecipitation to identify protein interaction partners

  • Phosphorylation status analysis for signaling pathway components

• Secretory pathway investigation:

  • Unfolded protein response (UPR) gene expression analysis

  • Comparison with specialized secretory cells like plasma cells

  • Assessment of rough ER structure via transmission electron microscopy

• Functional validation:

  • In vivo models testing myeloid cell production upon inflammatory challenge

  • In vitro differentiation assays with isolated MPP3 subpopulations

  • Cytokine blockade experiments to identify key mediators

These methodologies collectively enable researchers to dissect the molecular underpinnings of MPP3 function, particularly the distinct roles of ER high/FcγR+ versus ER low/FcγR− subpopulations.

What are the optimal storage and handling conditions for MPP3 antibodies?

For optimal performance of MPP3 antibodies, follow these storage and handling guidelines:

• Storage temperature:

  • Short-term (≤1 month): 2-8°C

  • Long-term: -20°C in small aliquots to avoid repeated freeze-thaw cycles

  • Avoid freezing at -80°C unless specifically recommended by the manufacturer

• Working dilutions:

  • Western blotting: Typically 1:500-1:2000 dilution

  • Immunofluorescence: Often 1:100-1:500 dilution

  • Flow cytometry: Usually 1:50-1:200 dilution

  • Optimization is recommended for each specific application and antibody

• Buffer composition:

  • For dilution: PBS with 0.1% BSA, 0.05% sodium azide, and 0.1% Tween-20

  • For long-term storage: Add 50% glycerol to prevent freeze-thaw damage

• Stability considerations:

  • Avoid repeated freeze-thaw cycles (create single-use aliquots)

  • Protect conjugated antibodies from light exposure

  • Monitor for signs of degradation (loss of specificity, increased background)

• Quality control:

  • Include positive controls (e.g., cell lysates known to express MPP3)

  • Run negative controls to assess non-specific binding

  • Periodically validate antibody performance against fresh stock

Following these guidelines will help maintain antibody integrity and ensure consistent experimental results when working with MPP3 antibodies.

What validation methods should be employed when using a new MPP3 antibody?

When adopting a new MPP3 antibody for your research, implement these validation methods:

• Specificity validation:

  • Western blot using positive control lysates expressing MPP3

  • Peptide competition assay using the immunogen peptide

  • siRNA/shRNA knockdown of MPP3 to confirm signal reduction

  • Comparison with alternative antibodies targeting different epitopes

• Application-specific validation:

  • For Western blotting: Confirm band appears at expected molecular weight

  • For flow cytometry: Compare staining pattern with established markers

  • For immunofluorescence: Verify expected subcellular localization

  • For immunoprecipitation: Confirm pull-down of known interaction partners

• Cross-reactivity assessment:

  • Test antibody against samples from multiple species if cross-reactivity is claimed

  • Verify predicted reactivity percentages (e.g., 100% human/mouse/rat, 93% guinea pig/horse)

  • Compare staining patterns between species to confirm consistent detection

• Orthogonal validation:

  • Confirm antibody results using alternative detection methods

  • Compare protein detection with mRNA expression data

  • Correlation with functional assays specific to MPP3

• Reproducibility testing:

  • Assess lot-to-lot variation if using multiple antibody batches

  • Test across different sample preparation methods

  • Verify consistent results across multiple biological replicates

Thorough validation ensures reliable results and prevents misinterpretation of data when studying the complex biology of MPP3 in various experimental contexts.

How can MPP3 antibodies contribute to understanding hematopoietic stress responses?

MPP3 antibodies are instrumental in elucidating hematopoietic stress responses through these research applications:

• Identification of stress-responsive populations:

  • Flow cytometric identification of expanding MPP3 subsets during stress

  • Tracking the shift toward FcγR+ MPP3 during myeloid regeneration

  • Quantifying changes in the proportion of ER high versus ER low MPP3

• Characterization of emergency myelopoiesis:

  • Monitoring MPP3 expansion prior to GMP and myeloid cell expansion

  • Analyzing the secretory profile of MPP3 during inflammatory challenge

  • Assessing the amplification of myeloid cell production by FcγR+/ER high MPP3

• Mechanism investigation:

  • Analyzing the autocrine/paracrine effects of MPP3-secreted cytokines

  • Studying how IL-10 production by stimulated MPP3 regulates TNFα and IL-6 secretion

  • Investigating how secretory MPP3 reinforce myeloid differentiation trajectories

• Pathological applications:

  • Examining dysregulated MPP3 function in hematological disorders

  • Identifying alterations in MPP3 subpopulations in inflammatory diseases

  • Studying the role of MPP3 as a self-reinforcing amplification compartment in disease conditions

• Therapeutic target identification:

  • Using MPP3 antibodies to identify and isolate populations for functional studies

  • Developing approaches to modulate MPP3 secretory function

  • Testing interventions that affect the balance between MPP3 subpopulations

MPP3 antibodies thus provide crucial tools for understanding how hematopoietic stem and progenitor cells respond to physiological challenges and contribute to emergency myelopoiesis.

What are the considerations for using MPP3 antibodies in multiparameter flow cytometry?

When designing multiparameter flow cytometry panels including MPP3 antibodies, consider these technical aspects:

• Panel design strategy:

  • Core markers for MPP3 identification: Lin−/c-Kit+/Sca-1+/Flk2−/CD48+/CD150−

  • Additional markers for subpopulation identification: FcγR, ER-Tracker dye

  • Select fluorophores based on expression level (brighter fluorophores for low-expressed markers)

• Fluorophore selection considerations:

  • Avoid spectral overlap between critical markers

  • Reserve brightest fluorophores (PE, APC) for key discriminating markers

  • Consider tandem dyes (PE-Cy7, APC-Cy7) for increased parameters

• Controls and compensation:

  • Single-stained controls for each fluorophore

  • Fluorescence-minus-one (FMO) controls for accurate gating

  • Biological controls (known positive and negative populations)

• Sample preparation optimization:

  • Standardize isolation procedures to minimize variability

  • Use viability dyes to exclude dead cells

  • Optimize fixation protocols if intracellular staining is required

• Critical gating strategy:

  • Sequential gating to identify MPP3 (Lin−/c-Kit+/Sca-1+/Flk2−/CD48+/CD150−)

  • Secondary gating for FcγR expression and ER-Tracker intensity

  • Approximately 31.0 ± 10.4% of MPP3 should be identified as ER high at steady state

• Analysis considerations:

  • Consider dimensionality reduction techniques (tSNE, UMAP) for visualization

  • Implement consistent batch analysis for longitudinal studies

  • Correlate flow cytometry findings with functional and molecular data

Proper implementation of these considerations ensures accurate identification and characterization of MPP3 subpopulations in complex experimental settings.

How can I integrate MPP3 antibody data with single-cell RNA sequencing results?

Integrating MPP3 antibody data with single-cell RNA sequencing requires a coordinated analytical approach:

• Experimental design integration:

  • FACS-sort MPP3 subpopulations (FcγR+/ER high vs. FcγR−/ER low) prior to scRNA-seq

  • Perform stimulation experiments (±LPS/Pam3CSK4) on sorted populations

  • Implement cell hashing or other multiplexing strategies to reduce batch effects

• Computational integration approaches:

  • Harmonize data by nearest neighbor integration methods

  • Implement uniform manifold approximation and projection (UMAP) for visualization

  • Apply batch correction algorithms to integrate data from multiple experiments

• Analytical strategies:

  • Use HSC and GMP conserved gene signature lists extracted from independent scRNA-seq analyses

  • Categorize clusters into functional groups (HSC gene-enriched immature, GMP gene-enriched myeloid-primed, metabolically activated intermediate)

  • Perform Gene Ontology (GO) and Slingshot analyses for cluster annotation and trajectory prediction

• Validation frameworks:

  • Confirm scRNA-seq clusters match flow cytometry-defined populations

  • Verify Fcgr3 expression in ER high MPP3 and immature HSC genes (Gata2, Meis1) in ER low MPP3

  • Use differentially expressed gene (DEG) analyses to identify marker genes for each subpopulation

• Functional correlation:

  • Connect transcriptional profiles with secretory capacity

  • Link gene expression patterns to differentiation potential

  • Correlate unique cytokine signatures with transcriptional states

This integrated approach provides comprehensive insight into the molecular heterogeneity of MPP3 and connects antibody-defined cellular phenotypes with underlying transcriptional programs.

What emerging technologies will enhance MPP3 antibody applications in research?

Several emerging technologies are poised to significantly advance MPP3 antibody applications:

• Spatial transcriptomics integration:

  • Combining MPP3 antibody staining with spatial transcriptomics to map MPP3 subpopulations within bone marrow niches

  • Correlating spatial distribution with functional heterogeneity

  • Investigating microenvironmental factors influencing MPP3 differentiation trajectories

• Mass cytometry (CyTOF) applications:

  • Developing MPP3 antibodies conjugated to rare earth metals

  • Creating high-dimensional panels (30+ parameters) to deeply phenotype MPP3 subsets

  • Integrating with signaling pathway analysis using phospho-specific antibodies

• Live-cell imaging innovations:

  • Developing non-disruptive MPP3 antibody fragments for live-cell tracking

  • Implementing intravital microscopy to observe MPP3 behavior in native bone marrow

  • Tracking cytokine secretion in real-time with reporter systems

• Nanobody and single-domain antibody development:

  • Creating smaller MPP3-targeting antibody fragments for improved tissue penetration

  • Developing intrabodies for tracking MPP3 in living cells

  • Engineering bispecific constructs targeting MPP3 and functional markers simultaneously

• Multi-omics integration platforms:

  • Combining antibody-based cell sorting with proteomics, metabolomics, and epigenomics

  • Implementing CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) to simultaneously capture surface protein and transcript information

  • Developing computational frameworks for integrating antibody-based data with multiple omics layers

These technological advances will enable researchers to gain unprecedented insight into MPP3 biology, particularly the dynamic changes in MPP3 subpopulations during inflammatory responses and their role in controlling myeloid differentiation.

What are the unresolved questions regarding MPP3 function that antibody research could address?

Despite recent advances, several unresolved questions about MPP3 function could be addressed through innovative antibody-based research:

• Heterogeneity beyond current classifications:

  • Are there additional MPP3 subpopulations beyond the FcγR+/ER high and FcγR−/ER low dichotomy?

  • Do these populations show tissue-specific variations or disease-specific alterations?

  • How stable are these phenotypes, and can they interconvert under certain conditions?

• Niche interaction dynamics:

  • How do MPP3 subpopulations interact with specific bone marrow niches?

  • What cellular and molecular factors regulate the transition from ER low to ER high MPP3?

  • How does paracrine signaling from MPP3 reshape the bone marrow microenvironment?

• Disease relevance:

  • How are MPP3 populations altered in hematological malignancies?

  • Could MPP3 secretory dysfunction contribute to inflammatory disorders?

  • Are there disease-specific MPP3 signatures that could serve as biomarkers?

• Developmental origins:

  • What are the developmental pathways leading to FcγR+/ER high versus FcγR−/ER low MPP3?

  • How is MPP3 heterogeneity established during ontogeny?

  • Are there epigenetic mechanisms maintaining MPP3 subpopulation identity?

• Therapeutic targeting potential:

  • Can MPP3 secretory function be selectively modulated for therapeutic benefit?

  • Would targeting specific MPP3 subpopulations affect emergency myelopoiesis without disrupting homeostatic hematopoiesis?

  • Could MPP3-directed therapies be developed for inflammatory disorders or malignancies?

Addressing these questions through antibody-based research approaches would significantly advance our understanding of hematopoietic regulation and potentially unveil new therapeutic strategies for various disorders.