CD163L1 Antibody

What is CD163L1 and what is its role in macrophage biology?

CD163L1 (CD163 molecule-like 1) is a 159.2 kDa protein belonging to the scavenger receptor cysteine-rich (SRCR) superfamily. It contains 12 SRCR domains and undergoes alternative splicing to produce multiple isoforms . CD163L1 serves as a critical marker distinguishing anti-inflammatory macrophages from pro-inflammatory subtypes. Studies have shown that CD163L1 expression is associated with tissue-resident macrophages exhibiting anti-inflammatory phenotypes, while its absence correlates with pro-inflammatory macrophage populations .

CD163L1 expression increases when monocytes differentiate into macrophages under M-CSF stimulation. This expression is further enhanced by anti-inflammatory mediators like IL-6 and IL-10, while being suppressed by pro-inflammatory mediators including IL-4, IL-13, TNF-α, and LPS/IFN-γ . Functionally, CD163L1 acts as an endocytic receptor that internalizes independently of cross-linking through a clathrin-mediated pathway .

How does CD163L1 differ from CD163 in experimental settings?

While CD163L1 arose through duplication of the CD163 gene in late evolution, these proteins have important experimental distinctions:

Despite structural similarities, CD163L1 exhibits clear differences in ligand preferences and tissue distribution .

Which applications are validated for CD163L1 antibodies?

Based on multiple commercial sources and research publications, CD163L1 antibodies have been validated for:

• Immunohistochemistry-paraffin (IHC-P): Most widely validated application

• Western Blot (WB): Validated with human samples

• ELISA: Validated for human CD163L1 detection

• Immunofluorescence (IF): Both cell culture and paraffin-embedded samples

When selecting an antibody, researchers should verify validation data for their specific application. For instance, anti-CD163L1 antibody [EPR6539] has been cited in multiple publications and shows strong validation for IHC-P applications with human samples .

How does CD163L1 expression change in inflammatory disease contexts?

CD163L1 expression shows distinct patterns in healthy versus inflammatory conditions, making it valuable for studying disease mechanisms:

In healthy tissues:

• CD163L1+ macrophages are predominant in liver and colon tissue

• These cells are typically CD14−CD209+CD11b−CD11c−TNF−IL-10+

• They exhibit high IL-10 production and CD209 expression

In inflammatory bowel disease (IBD):

• CD163L1+ macrophages lose their ability to produce IL-10 and express CD209

• CLEC5A+ macrophages become abundant in the intestinal lamina propria

• Higher numbers of CLEC5A+CD163L1+ double-positive cells appear compared to healthy tissues

In melanoma:

• CD163L1+ tumor-associated macrophages (TAMs) are found in 100% of cases

• CLEC5A+ TAMs are absent in 42% of cases

• CD163L1+ TAMs express high levels of CD209 and produce significant IL-10

• In metastases, CLEC5A+ TAMs are CD14hi and produce enhanced TNF levels

This differential expression makes CD163L1 antibodies valuable tools for studying macrophage phenotype shifts during disease progression.

What epitope considerations are important for CD163L1 antibody selection?

The choice of epitope target affects experimental outcomes with CD163L1 antibodies:

• N-terminal vs. internal epitopes: Antibodies targeting N-terminal regions may not detect all isoforms, as alternative splicing can affect these regions . Antibodies like those targeting the N-terminal (ab227300) are optimal for Western blot applications but may have limitations in certain contexts .

• Conformational vs. linear epitopes: Some CD163L1 mAbs (like 6E8 and 9A10) recognize conformational epitopes rather than linear ones. For example, neither 6E8 nor 9A10 react with CD163 SRCR 5-9 in Western blot, suggesting they recognize three-dimensional structures .

• Immunogen sequence: When available, examine the specific immunogen sequence. For example, one antibody (HPA015663) utilizes an immunogen sequence of "LRVSTRRRGSLEENLFHEMETCLKREDPHGTRTSDDTPNHGCEDASDTSLLGVLPASEAT" .

For highest specificity when targeting different isoforms, consider using antibodies generated against recombinant fragments of human CD163L1 protein that recognize specific domains.

What are optimal sample preparation methods for CD163L1 detection in tissues?

For optimal CD163L1 detection in tissue samples:

Fixation:

• Formalin-fixed paraffin-embedded (FFPE) tissues show good results with CD163L1 antibodies

• Fixation time should be optimized as overfixation can mask epitopes

Antigen Retrieval:

• Heat-induced epitope retrieval using Tris-EDTA buffer (pH 9.0) is recommended

• Citrate buffer (pH 6.0) may be less effective for certain antibody clones

Antibody Dilutions:

• For IHC-P: Typical dilutions range from 1:200-1:2000 depending on the antibody

• For recombinant monoclonal antibodies like EPR6539: 1:2000 dilution is effective

• For polyclonal antibodies: 1:200-1:500 is often suitable

Visualization:

• Secondary antibodies conjugated with HRP and DAB detection system work effectively

• For fluorescence detection, TSA (tyramide signal amplification) can improve sensitivity for low-expression samples

Always include appropriate positive controls (human spleen or lymphoid tissue) and negative controls (primary antibody omission) in your experimental design.

How can I validate the specificity of CD163L1 antibodies for my research?

A comprehensive validation strategy should include:

• Positive and negative tissue controls:

  • Positive: Human spleen, lymphoid tissue, and colon tissues are recommended

  • Negative: Tissues known to lack CD163L1 expression

• Knockdown/knockout validation:

  • siRNA or CRISPR/Cas9-mediated CD163L1 knockdown/knockout in relevant cell lines

  • Compare staining in wild-type vs. knockdown/knockout samples

• Orthogonal validation:

  • Compare protein detection with mRNA expression (qPCR or RNA-Seq)

  • Some antibodies (like HPA015663) feature enhanced validation with orthogonal RNAseq

• Independent antibody validation:

  • Compare results from antibodies targeting different epitopes

  • For example, compare results between N-terminal antibodies and those targeting internal domains

• Recombinant protein blocking:

  • Pre-incubate antibody with recombinant CD163L1 protein before application

  • Should abolish specific staining in positive samples

Detailed documentation of validation experiments increases confidence in research findings and should be included in publications.

What methodological approaches can distinguish between CD163L1 isoforms?

CD163L1 undergoes alternative splicing to produce up to 4 different isoforms with different subcellular localizations and functions . To distinguish between these isoforms:

• Isoform-specific antibodies:

  • Select antibodies raised against unique regions of specific isoforms

  • For membrane-bound isoforms (1 and 2), target the transmembrane region

  • For secreted isoform (3), target unique C-terminal sequences

• Subcellular fractionation:

  • Separate membrane fractions from cytosolic and secreted proteins

  • Analyze fractions by Western blot to differentiate membrane-bound from secreted isoforms

• Molecular weight discrimination:

  • Use high-resolution SDS-PAGE to separate proteins based on size

  • Isoform 1 is the full-length protein (159.2 kDa)

  • Isoform 2 is exclusively found in spleen

  • Isoform 3 is a secreted variant and should be detectable in culture media

• RNA analysis:

  • Design PCR primers or probes that span exon-exon junctions unique to each isoform

  • Correlate protein detection with isoform-specific mRNA expression

Research has shown that two cytoplasmic splice variants of CD163L1 are differentially expressed and have different subcellular distribution patterns , making isoform-specific detection methodologically important.

How do CD163L1 antibodies perform in multi-parameter flow cytometry?

While IHC-P is the most validated application, researchers interested in multi-parameter flow cytometry should consider:

• Fluorophore conjugation:

  • FITC-conjugated antibodies (NBP3-24128F) are available for direct fluorescence detection

  • For unconjugated primary antibodies, select secondary antibodies with minimal spectral overlap with other markers

• Surface vs. intracellular staining:

  • CD163L1 can be detected on the cell surface (isoforms 1 and 2)

  • For complete detection including cytoplasmic pools, permeabilization is required

  • Recommended protocol: Fix cells with 4% paraformaldehyde followed by permeabilization with 0.1% saponin

• Gating strategy for macrophage subpopulations:

  • First gate on CD68+ cells to identify macrophage populations

  • Then examine CD163 and CD163L1 expression

  • Further characterize using markers like CLEC5A, CD14, CD209, CD11b, CD11c

  • Analyze cytokine production (TNF, IL-10) in different subsets

• Panel design considerations:

  • CD163L1 expression strongly correlates with CD163, but shows distinct patterns in certain macrophage subsets

  • Include markers that distinguish inflammatory (CLEC5A+) from anti-inflammatory (CD163L1+) macrophages

Research has demonstrated that CD163L1+ macrophages can be clearly distinguished from CLEC5A+ inflammatory macrophages by flow cytometry in various tissue contexts .

What are the latest methodological advances in studying CD163L1 function?

Recent methodological advances have expanded our understanding of CD163L1 function:

• Receptor-ligand interaction studies:

  • Native nESI-qTOF MS has been employed to analyze glycoengineered antibody interactions with related receptors

  • Similar approaches could elucidate CD163L1 binding partners

• Endocytosis assays:

  • CD163L1 functions as an endocytic receptor that internalizes through a clathrin-mediated pathway

  • Clathrin-dependent endocytosis can be studied using inhibitors like chlorpromazine or siRNA against clathrin

• Resolution of inflammation models:

  • CD163L1 likely has roles in resolving inflammation

  • In vitro models using primary macrophages treated with pro-resolving mediators can help elucidate these functions

  • Cytokine secretion profiles should be analyzed following CD163L1 engagement

• Macrophage polarization studies:

  • CD163L1 expression increases when monocytes differentiate into anti-inflammatory macrophages

  • Flow cytometry panels including CD163L1 can track macrophage polarization dynamics

  • Combined with functional assays (phagocytosis, cytokine production) to correlate expression with function

By incorporating these methodological approaches, researchers can move beyond descriptive studies to understand the functional significance of CD163L1 in health and disease.

How should I address weak or absent CD163L1 staining in IHC applications?

If experiencing weak or absent CD163L1 staining in IHC:

• Optimize antigen retrieval:

  • Test different methods: Heat-induced epitope retrieval using Tris-EDTA buffer (pH 9.0) is recommended for most CD163L1 antibodies

  • Extend retrieval time (15-30 minutes) if initial results are weak

• Adjust antibody concentration:

  • Increase antibody concentration (decrease dilution)

  • For monoclonal antibodies like EPR6539, try 1:1000 instead of 1:2000

  • For polyclonal antibodies, try 1:100-1:200 instead of 1:500

• Extend incubation time:

  • Incubate primary antibody overnight at 4°C instead of 1 hour at room temperature

  • Use humidity chamber to prevent evaporation

• Enhance signal amplification:

  • Implement polymer-based detection systems for higher sensitivity

  • Consider tyramide signal amplification for fluorescence applications

• Verify tissue expression:

  • CD163L1 expression varies by tissue type; confirm your sample should express the protein

  • Use human spleen or lymphoid tissue as positive controls

  • Remember that alveolar macrophages, glia, and Kupffer cells show low or absent expression

How can I minimize background in CD163L1 immunostaining?

High background can obscure specific CD163L1 staining. Address this issue by:

• Blocking optimization:

  • Extend blocking time (60 minutes)

  • Test different blocking agents (5% BSA, 10% normal serum, commercial blocking solutions)

  • Add 0.1-0.3% Triton X-100 to blocking solution for better penetration

• Antibody dilution optimization:

  • Increase antibody dilution (1:500 to 1:1000)

  • Prepare antibodies in blocking solution rather than plain buffer

• Washing steps:

  • Increase number and duration of washing steps

  • Use agitation during washing

  • Add 0.05-0.1% Tween-20 to wash buffer

• Endogenous enzyme blocking:

  • Block endogenous peroxidase with 3% H₂O₂ for 10 minutes

  • For alkaline phosphatase detection, use levamisole to block endogenous activity

• Fluorescence considerations:

  • Use Sudan Black B (0.1-0.3%) to reduce autofluorescence

  • Include antifade reagents in mounting medium

• Antibody specificity:

  • Consider using monoclonal antibodies which typically have lower background

  • Affinity-purified antibodies like HPA015663 can provide cleaner results

What controls are essential when publishing research using CD163L1 antibodies?

For rigorous publication standards, include these controls:

• Positive tissue controls:

  • Human spleen, lymph nodes, or colon tissues known to express CD163L1

  • CD163L1 is highly expressed in spleen, rectum, lymph node, colon, and appendix

• Negative tissue controls:

  • Tissues known to lack or have minimal CD163L1 expression

  • Alveolar macrophages show low/absent expression

• Technical controls:

  • Isotype controls: Use matched isotype (IgG) at equivalent concentration

  • Secondary antibody only: Omit primary antibody to assess non-specific binding

  • Absorption controls: Pre-incubate antibody with recombinant antigen

• Biological validation:

  • Correlation with CD163L1 mRNA expression

  • Comparison with other established macrophage markers (CD68, CD163)

  • Known biological relationships (e.g., increased expression with IL-10 treatment)

• Multi-antibody validation:

  • Use two different antibodies targeting distinct epitopes

  • For example, combine the results from antibody clones CD163L1/7971 and CD163L1/7972

How can CD163L1 antibodies be used in studying macrophage polarization dynamics?

CD163L1 antibodies offer valuable tools for investigating macrophage polarization:

• Temporal analysis of macrophage differentiation:

  • Track CD163L1 expression as monocytes differentiate into macrophages

  • CD163L1 expression increases when monocytes are M-CSF stimulated to macrophages

  • Combine with markers like CLEC5A to distinguish pro- vs. anti-inflammatory phenotypes

• Cytokine response studies:

  • CD163L1 expression increases with IL-6 and IL-10 stimulation

  • Expression is suppressed by IL-4, IL-13, TNF-α, and LPS/IFN-γ

  • Use CD163L1 antibodies to track polarization shifts in response to cytokine treatment

• Disease progression models:

  • In inflammatory bowel disease, monitor shifts from CD163L1+ anti-inflammatory macrophages to CLEC5A+ inflammatory types

  • In cancer, track tumor-associated macrophage phenotypes, as CD163L1+ TAMs produce significant IL-10

• Therapeutic intervention assessment:

  • Evaluate how macrophage-targeting therapies affect CD163L1 expression

  • Correlate changes in CD163L1+ macrophage populations with clinical outcomes

Researchers can develop multi-parameter panels that include CD163L1 alongside other polarization markers to obtain a comprehensive view of macrophage heterogeneity in health and disease.

What is the potential of CD163L1 as a biomarker in inflammatory diseases?

CD163L1 shows promise as a biomarker in several contexts:

• Inflammatory bowel disease:

  • CD163L1+ macrophages in healthy colon produce IL-10

  • In IBD, these cells lose IL-10 production capacity and CD209 expression

  • The ratio of CLEC5A+ to CD163L1+ macrophages increases in active disease

• Cancer immunology:

  • CD163L1+ tumor-associated macrophages are found in 100% of melanoma cases

  • The balance between CD163L1+ (anti-inflammatory) and CLEC5A+ (pro-inflammatory) TAMs may predict tumor progression

• Resolution of inflammation:

  • CD163L1 likely functions as a scavenger receptor for ligands involved in resolution of inflammation

  • Changes in CD163L1 expression may mark transition from inflammatory to resolution phases

• Tissue-specific macrophage phenotyping:

  • CD163L1 expression varies across tissue macrophage populations

  • May help identify tissue-specific macrophage subsets with distinct functional properties

Future research should explore correlations between CD163L1+ macrophage populations and clinical outcomes in various inflammatory conditions, potentially establishing this marker as a diagnostic or prognostic tool.

What emerging technologies complement CD163L1 antibody-based research?

Cutting-edge technologies that enhance CD163L1 research include:

• Single-cell technologies:

  • Single-cell RNA-seq to correlate CD163L1 protein expression with transcriptional profiles

  • Mass cytometry (CyTOF) for high-dimensional analysis of CD163L1+ cells

  • Imaging mass cytometry for spatial context of CD163L1+ macrophages in tissues

• Spatial biology approaches:

  • Multiplex immunofluorescence to visualize CD163L1+ macrophages in relation to other cell types

  • Spatial transcriptomics to map CD163L1 expression patterns within tissue microenvironments

  • Combined with artificial intelligence for pattern recognition across large tissue areas

• Functional genomics:

  • CRISPR/Cas9 editing of CD163L1 to elucidate functional roles

  • ChIP-seq to identify transcription factors regulating CD163L1 expression

  • Epigenetic profiling to understand regulation of CD163L1 in different macrophage subsets

• Advanced imaging:

  • Live-cell imaging with fluorescently-tagged CD163L1 antibodies to track endocytosis

  • Super-resolution microscopy for nanoscale localization within membrane microdomains

  • Intravital microscopy to observe CD163L1+ macrophages in vivo