CD164 Antibody

What is CD164 and what are its main functions in hematopoiesis?

CD164 is a transmembrane sialomucin that functions as both an adhesion receptor on human CD34+ cell subsets in bone marrow and as a potent negative regulator of CD34+ hemopoietic progenitor cell proliferation. These diverse effects are mediated by at least two functional epitopes defined by specific monoclonal antibodies . CD164 plays crucial roles in regulating the adhesion of CD34+ cells to bone marrow stroma and inhibiting the proliferation and differentiation of primitive CD34+ erythroid and granulocyte-monocyte progenitors in colony forming assays . Additionally, CD164 prevents the recruitment of CD34+CD38low/− cells into cycle in response to cytokines such as IL-3, IL-6, stem cell factor (SCF), and G-CSF . These findings suggest that CD164 acts as a potent negative signaling molecule for hematopoietic progenitor cell proliferation.

What is the molecular structure of CD164?

CD164 is a single pass transmembrane protein with a short cytoplasmic tail that is highly N- and O-glycosylated . The protein's extracellular region consists of two O-glycosylated mucin domains (I and II) that are linked by a non-mucin cysteine-rich subdomain . CD164 contains sialic acid and a Ser-Gly motif that may serve as an attachment site for glycosaminoglycan side chains . The molecule typically forms a homodimer with an apparent molecular weight of 160 kDa, while the monomeric form runs at 80-100 kDa on SDS-PAGE . Three splice variants of CD164 have been reported with apparent molecular weights ranging between 80-100 kDa .

What are the main antibody clones available for CD164 research?

Several well-characterized monoclonal antibodies against CD164 are available for research:

The 67D2 monoclonal antibody is particularly versatile, recognizing human CD164 (also known as sialomucin CD164, MUC-24, and multi-glycosylated core protein 24) with applications in flow cytometry, Western blotting, and immunofluorescence .

How do the epitopes recognized by different CD164 antibody clones differ?

CD164 antibody clones recognize distinct epitopes on the molecule, which has important implications for their applications in research. Using CD164 splice variants and soluble recombinant chimeric proteins, researchers have mapped these epitopes with precision :

• The 105A5 and 103B2/9E10 functional epitopes map to distinct glycosylated regions within the first mucin domain of CD164 .

• The N6B6 and 67D2 mAbs recognize closely associated complex epitopes that rely on the conformational integrity of the CD164 molecule and encompass the cysteine-rich regions encoded by exons 2 and 3 .

Based on their sensitivities to enzymatic treatments, CD164 epitopes have been grouped into three classes :

• Class I (105A5): Sensitive to sialidase, O-glycosidase, and O-sialoglycoprotease

• Class II (103B2/9E10): Sensitive to N-glycanase, O-glycosidase, and O-sialoglycoprotease

• Class III (N6B6 and 67D2): Not removed by such enzyme treatments

This classification is analogous to CD34 epitope classification and provides important insights into the structure-function relationships of CD164 .

What cell types express CD164 and at what levels?

CD164 expression varies across different cell types and developmental stages:

• Highest expression: The most primitive hematopoietic progenitors (CD34high, AC133high, CD38low) show the highest cell surface expression of CD164 .

• Hematopoietic cells: CD164 is expressed on CD34+ hematopoietic cells, myeloid and erythroid progenitors, and activated basophils .

• Bone marrow: Expression is found on bone marrow stromal cells and on the vast majority of lin−CD34low/−CD38low/− cells with capacity for long-term repopulation of hematopoiesis .

• Mature blood cells: Low or negligible levels of expression on peripheral blood neutrophils and erythrocytes .

• Non-hematopoietic tissues: Expression has been reported in various carcinomas and leukemic cells, and in tissues including the small intestine, colon, lung, and thyroid .

Interestingly, CD164 epitopes defined by different antibodies are differentially and often reciprocally expressed on lymphoid cells, endothelia, postcapillary high endothelial venules, and basal/subcapsular epithelia in hematopoietic and nonhematopoietic tissues .

What are the optimal protocols for using CD164 antibodies in flow cytometry?

For optimal flow cytometry detection of CD164, follow these guidelines:

Sample Preparation:

• Isolate mononuclear cells from bone marrow, cord blood, or peripheral blood using density gradient centrifugation

• Wash cells twice in phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS)

• Adjust cell concentration to 1-5 × 10^6 cells/ml

Staining Procedure:

• Aliquot 100 μl of cell suspension (1-5 × 10^5 cells) into flow cytometry tubes

• Add appropriate amount of CD164 antibody (typically 5-10 μg/ml for purified antibodies like 67D2 or according to manufacturer's recommendation for conjugated antibodies)

• Include appropriate isotype controls (e.g., mouse IgG1, κ for 67D2 clone)

• Incubate for 30 minutes at 4°C in the dark

• For unconjugated primary antibodies, wash cells twice with PBS/2% FBS and add fluorochrome-conjugated secondary antibody

• For multicolor analysis, add other directly conjugated antibodies (e.g., anti-CD34-FITC, anti-CD38-PE-Cy7)

• Wash cells twice with PBS/2% FBS

• Resuspend cells in 300-500 μl of PBS containing 2% FBS and a viability dye

Analysis Considerations:

• For CD164+ cell identification, first gate on viable cells, then on CD34+ population, and finally analyze CD164 expression

• The 67D2 clone has been shown to be useful for flow cytometric detection of human CD164

• When examining CD164 expression in primitive hematopoietic progenitors, analyze in conjunction with CD34, CD38, and other relevant markers

How can researchers optimize Western blotting for CD164 detection?

Western blotting for CD164 requires careful consideration of several factors:

Sample Preparation:

• Use appropriate lysis buffers containing detergents (e.g., 1% Triton X-100 or 4× modified Laemmli buffer with 0.5% SDS) and 1× Complete protease inhibitors

• Maintain sample integrity by keeping samples cold and adding protease inhibitors

Gel Electrophoresis:

• Use 6-10% SDS-PAGE gels to properly resolve CD164 (80-100 kDa)

• Run samples under both reducing and non-reducing conditions for comprehensive analysis

Transfer and Detection:

• Use low methanol concentration in transfer buffer for efficient transfer of high molecular weight glycoproteins

• Block membranes adequately to reduce background (5% non-fat milk or BSA)

• The 67D2 antibody has been validated for Western blotting under non-reducing conditions, detecting an 80-100 kDa protein as well as a high molecular weight aggregate of approximately 220 kDa

Important Considerations:

• Different cell types may show variable electrophoretic mobility of CD164 due to differences in glycosylation

• Promonocytic cell line THP-1 and myelomonocytic cell line HL60 exhibit lower electrophoretic mobilities compared to other cell lines

• For definitive analysis, consider using multiple antibodies recognizing different epitopes

What approaches can be used for validating CD164 antibody specificity?

Validating CD164 antibody specificity requires a multi-faceted approach:

Genetic Validation:

• Use CD164 knockdown/knockout models (siRNA, shRNA, or CRISPR-Cas9)

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

• Test antibodies on CD164 transfection models (e.g., MS.5 mouse stromal cells transfected with CD164 cDNA constructs)

Biochemical Validation:

• Test antibody reactivity on recombinant CD164 proteins and different CD164 splice variants

• Perform epitope-specific validation using enzymatic treatments:

  • For class I epitopes (105A5): Test with sialidase, O-glycosidase, and O-sialoglycoprotease

  • For class II epitopes (103B2/9E10): Test with N-glycanase, O-glycosidase, and O-sialoglycoprotease

  • For class III epitopes (N6B6, 67D2): Confirm resistance to such enzyme treatments

• Use peptide blocking to confirm specificity

Cross-Validation:

• Compare staining patterns using multiple CD164 antibody clones

• Perform IP with one CD164 antibody clone and Western blot with another

• Use appropriate cell line controls (e.g., KG1a cells for positive control)

How can CD164 antibodies be used for functional studies of hematopoietic progenitors?

CD164 antibodies, particularly the 103B2/9E10 and 105A5 clones, have been used effectively for functional studies of hematopoietic progenitors:

Adhesion Studies:

• The 103B2/9E10 mAb inhibits the adhesion of CD34+ cells to bone marrow stromal cells in vitro

• This enables researchers to study the role of CD164 in hematopoietic progenitor cell-microenvironment interactions

Proliferation and Differentiation Studies:

• Both 103B2/9E10 and 105A5 mAbs inhibit nucleated cell production in liquid cultures and colony formation by primitive granulocyte-monocyte and erythroid precursors in clonogenic assays from CD34+ cells

• This allows investigation of CD164's role in regulating hematopoietic progenitor cell proliferation and differentiation

Cell Cycle Regulation:

• The 103B2/9E10 mAb prevents recruitment of CD34+CD38low/− cells into cycle in the presence of IL-3, IL-6, G-CSF, and SCF

• This enables studies of how CD164 influences cell cycle entry and progression in primitive hematopoietic cells

For these functional studies, researchers should:

• Use purified antibody preparations free of preservatives and endotoxins

• Perform dose-response experiments (typically 1-50 μg/ml)

• Include appropriate isotype-matched control antibodies

• Consider the timing of antibody addition (pre-incubation vs. continuous presence)

How can researchers distinguish between different CD164 splice variants?

Distinguishing between CD164 splice variants requires a combined approach:

Splice Variant Characteristics:

• Several CD164 splice variants have been identified, including full-length CD164(E1-6), CD164(EΔ4) lacking exon 4, and CD164(EΔ5) lacking exon 5

• These variants can be generated using PCR amplification with appropriate primers targeting specific exons

Experimental Approaches:

• Molecular Analysis:

  • RT-PCR with primers specific for different exons

  • CD164 isoforms can be PCR amplified from templates subcloned in appropriate vectors after RT-PCR analyses

• Expression System Analysis:

  • Generate transfectants expressing individual CD164 splice variants

  • CD164 splice variant cDNAs can be transfected into cell lines (e.g., MS.5 mouse stromal cells) using calcium phosphate as a facilitator

  • Compare the reactivity of different antibodies with each variant

• Western Blotting:

  • Use antibodies recognizing different domains of CD164

  • Compare banding patterns and molecular weights

  • Transiently transfected cells can be lysed directly in modified Laemmli reducing sample buffer and resolved by 6 or 10% SDS-PAGE before immunoblotting

• Immunocytochemistry:

  • Transiently transfected cells can be fixed in situ and stained with each CD164 mAb or with an irrelevant isotype-matched control mAb

  • This allows visualization of expression patterns of different variants

What challenges arise from post-translational modifications of CD164?

CD164's extensive post-translational modifications present several challenges for researchers:

Glycosylation Heterogeneity:

• CD164 is heavily glycosylated with both N-linked and O-linked carbohydrates

• Glycosylation patterns vary between different cell types, resulting in molecular weight variations (80-100 kDa range)

• This heterogeneity can affect antibody binding efficiency and specificity

Epitope Accessibility:

• Different CD164 epitopes show varying sensitivities to glycosylation:

  • Class I epitopes (105A5): Sensitive to sialidase, O-glycosidase, and O-sialoglycoprotease

  • Class II epitopes (103B2/9E10): Sensitive to N-glycanase, O-glycosidase, and O-sialoglycoprotease

  • Class III epitopes (N6B6, 67D2): Not removed by such enzyme treatments

Solutions:

• Use multiple antibodies recognizing different epitope classes for comprehensive analysis

• For consistent detection regardless of glycosylation status, prefer class III epitope antibodies (N6B6, 67D2)

• Consider enzymatic deglycosylation treatments for comparative studies:

  • Sialidase treatment: Removes sialic acid residues

  • O-sialoglycoprotease treatment: Cleaves O-glycosylated proteins

  • N-glycanase treatment: Removes N-linked glycans

  • O-glycosidase treatment: Removes O-linked glycans

How can researchers troubleshoot inconsistent CD164 antibody staining?

When facing inconsistent CD164 antibody staining, consider these troubleshooting approaches:

Flow Cytometry Issues:

• Weak or No Signal:

  • Check antibody viability and concentration

  • Verify that your cells express CD164 (use KG1a cells as positive control)

  • Consider using antibodies against class III epitopes (67D2, N6B6) which are less affected by glycosylation

  • Optimize staining conditions (temperature, time, buffer)

• High Background:

  • Include proper blocking steps (Fc block)

  • Titrate antibody to optimal concentration

  • Use appropriate isotype controls (mouse IgG1, κ for 67D2 and N6B6 clones)

Western Blotting Challenges:

• Multiple Bands:

  • CD164 can appear as monomeric form (80-100 kDa) and high molecular weight aggregate (~220 kDa)

  • Different glycoforms may appear as bands of different molecular weights

  • Try both reducing and non-reducing conditions

• No Signal:

  • Verify transfer efficiency

  • Consider using class III epitope antibodies (67D2) which are less affected by denaturation

  • Test different antibody concentrations (1:500-1:2000 dilution range)

Immunohistochemistry Solutions:

• Use appropriate antigen retrieval:

  • TE buffer pH 9.0 is recommended as the primary choice

  • Citrate buffer pH 6.0 can be used as an alternative

• Optimize antibody dilution (1:20-1:200 for IHC)

• Consider tissue-specific factors that may affect CD164 detection

How can CD164 antibodies be integrated into multiparametric analysis of hematopoietic cells?

CD164 antibodies can be effectively incorporated into multiparametric analysis for comprehensive characterization of hematopoietic cells:

Key Marker Combinations:

• CD164 + CD34: Identifies primitive hematopoietic progenitors, with the highest CD164 expression on CD34high cells

• CD164 + CD34 + CD38: Further refines identification of primitive cells (CD34+CD38low/- with high CD164 expression)

• CD164 + CD34 + AC133 (CD133): Identifies very early progenitors (CD34high, AC133high, CD164high)

• Extended panels can include lineage markers, CD90, CD45RA, and CD49f for comprehensive HSC characterization

Panel Design Considerations:

• Choose bright fluorochromes (e.g., PE, APC) for CD164 and other markers expressed at lower levels

• Ensure proper compensation when using multiple fluorochromes

• Include appropriate controls (FMO controls, isotype controls)

• Consider using multiple CD164 antibodies recognizing different epitopes for more comprehensive analysis

Analysis Approaches:

• Conventional gating: First gate on viable cells, then lin- population, followed by CD34+ cells, and finally analyze CD164 expression in conjunction with other markers

• Advanced computational analysis: Consider high-dimensional data visualization tools (viSNE, t-SNE, UMAP) and clustering algorithms for identifying complex cell populations

What are emerging applications of CD164 antibodies in cancer research?

CD164 antibodies are finding increasing applications in cancer research:

Expression in Malignancies:

• CD164 expression has been reported on various carcinomas and leukemic cells

• Elevated expression of CD164 has been observed in patients with Sézary syndrome, a primary cutaneous T-cell lymphoma, suggesting its potential as a marker of this disease

Cancer Cell Identification:

• The 67D2 antibody has been used to detect CD164 in various cancer cell lines, including:

  • BxPC-3 cells (pancreatic cancer)

  • U-87 MG cells (glioblastoma)

  • PC-3 cells (prostate cancer)

Functional Studies:

• CD164 has been reported to be involved in cancer metastasis

• Antibodies can be used to investigate the role of CD164 in cancer cell adhesion, migration, and proliferation

Technical Applications:

• Flow cytometry: Detection of CD164 in CXCL12-treated PC-3 human prostate cancer cell line has been demonstrated

• Immunohistochemistry: CD164 detection in human pancreatic cancer tissue has been validated

• Western blotting: Can be used to analyze CD164 expression levels in various cancer cell lines