Anti-Mouse CD90.2 (Thy 1.2) [Clone 30-H12] — Purified in vivo GOLD™ Functional Grade

Clone 30-H12, an anti-mouse CD90.2 (Thy1.2) monoclonal antibody, is used in in vivo mouse studies primarily to deplete T lymphocytes and as a cell surface marker for identifying specific cell populations such as hematopoietic stem cells and axonal processes of mature neurons.

Key in vivo uses include:

• T Cell Depletion: 30-H12 is widely deployed to selectively deplete T cells in mice expressing Thy1.2, facilitating studies on immune function, graft-versus-host disease, and immunodeficiency.
• Cell Population Identification: It serves as a marker in flow cytometry (FACS) to identify and sort thymocytes, peripheral T lymphocytes, hematopoietic stem cells, neurons, epithelial cells, and fibroblasts.
• Functional Manipulation: Crosslinking with this antibody can induce Ca2+ flux in thymocytes, modulate cell signaling, promote apoptosis, and inhibit T cell proliferation, making it useful for mechanistic studies of T lymphocyte function.
• Distinction between Mouse Strains: Used to distinguish Thy1.2 (CD90.2) expressing strains (e.g., C57BL/6, BALB/c) from Thy1.1 strains in adoptive transfer and transplantation research.

Additional details:

• 30-H12 does not cross-react with Thy1.1 (CD90.1), and thus is specific for the majority of mouse strains but not AKR/J or PL strains.
• It is often used in combination with other markers for multi-parameter cell sorting and stem cell research, and as a depletion tool in vivo administration, typically via intraperitoneal or intravenous injection dependent on the experimental design.

In summary, clone 30-H12's principal in vivo functions are immune cell depletion, identification and sorting of specific mouse cell populations, and modulation of T cell signaling and apoptosis.

The correct storage temperature for sterile packaged clone 30-H12 (Anti-Mouse CD90.2/Thy 1.2) is 2–8°C for short-term storage (up to 1 week), and for long-term storage, aliquot and store at –20°C in a manual defrost freezer.

• Short-term storage (?1 week): 2–8°C (refrigerator).
• Long-term storage: Aliquot without dilution and store at –20°C; avoid repeated freeze-thaw cycles.
• For best preservation, store the antibody undiluted and protect from prolonged light exposure if it is conjugated.

These recommendations are standard for purified monoclonal antibodies, including clone 30-H12. Always refer to the lot-specific datasheet included with your product, as vendor formulations or conjugates may require slightly different handling.

Commonly Used Antibodies and Proteins Paired with 30-H12

The 30-H12 antibody specifically recognizes mouse CD90.2 (Thy-1.2), a GPI-anchored glycoprotein expressed on thymocytes, mature T lymphocytes, hematopoietic stem cells, neurons, and some epithelial and fibroblast populations. In the literature, 30-H12 is frequently employed in multi-color flow cytometry panels, functional assays, and in vivo depletion studies. Here are some of the other commonly used antibodies and proteins paired with 30-H12 in various contexts:

Co-Staining and Flow Cytometry

• Anti-CD3/TCR complex antibodies (e.g., 145-2C11): Often used alongside 30-H12 to identify and characterize T cell populations, particularly in studies of T cell activation, costimulation, and intracellular signaling.
• Anti-CD4 and Anti-CD8: Used to further delineate helper (CD4+) and cytotoxic (CD8+) T cell subsets within the CD90.2-positive population.
• Anti-CD25 (IL-2 receptor alpha): Frequently combined with 30-H12 and CD4 to identify regulatory T cells (CD4+CD25+Foxp3+) in immunophenotyping panels.
• Anti-CD45: CD90.2 is reported to interact with CD45, a pan-leukocyte marker, in signal transduction pathways; hence, antibodies against CD45 are often used in concert with 30-H12 to study leukocyte subsets and signaling cascades.
• Anti-CD16/CD32 (Fc receptor blockers): Pre-treatment with these antibodies is recommended prior to immunofluorescent staining to reduce non-specific binding to cells bearing Fc receptors.
• Isotype controls (e.g., Rat IgG2b-PE for flow cytometry): Used as negative controls to set gating thresholds and verify staining specificity.

Functional Assays and In Vivo Applications

• Depletion antibodies: 30-H12 is frequently used for in vivo depletion of T cells, often in models of autoimmunity, transplantation, or infection. In these studies, 30-H12 may be used alone or in combination with other cell-depleting antibodies (e.g., anti-CD4, anti-CD8).
• Costimulatory molecules: In studies of T cell activation, 30-H12 may be used with antibodies against costimulatory molecules such as CD28 or CTLA-4 to investigate T cell signaling and function.
• Calcium flux assays: 30-H12 has been reported to induce Ca2+ flux in thymocytes, and these assays sometimes include reagents to measure calcium mobilization alongside other activation markers.

Immunohistochemistry and Microscopy

• Pan-cytokeratin or EpCAM (for epithelial cells): In studies of non-lymphoid tissues, 30-H12 may be combined with epithelial markers to distinguish lymphocytes from epithelial cells.
• Neural markers (e.g., NeuN, GFAP): For studies in the nervous system, 30-H12 (labeling neurons, glia, or neural precursors) is often paired with specific neural markers.
• Apoptosis markers (e.g., Annexin V, Caspase-3): Used in studies investigating the role of CD90.2 in cell survival or death pathways.

Multiplexed and High-Dimensional Techniques

• CODEX® and CyTOF®: In advanced multiplex imaging and cytometry platforms, 30-H12 is included in large antibody panels to enable high-dimensional cell phenotyping.
• PhenoCycler® (formerly CODEX): Used in spatial biology studies to co-localize CD90.2+ cells with other markers within tissue sections.

Summary Table

Key Points

• 30-H12 is a versatile reagent used across a wide range of applications, often in combination with lineage-specific, functional, and activation markers.
• The choice of paired antibodies depends on the biological question, ranging from basic immunophenotyping to advanced multiplex imaging and functional assays.
• Negative controls (isotype-matched antibodies) and Fc receptor blockers are essential for robust and specific staining, especially in multi-parameter flow cytometry.
• Interactions with CD45 highlight the importance of including pan-leukocyte markers in studies of CD90.2-mediated signaling.

In summary, 30-H12 is most commonly used with antibodies against CD3, CD4, CD8, CD25, CD45, and Fc receptor blockers in immunophenotyping, and with epithelial or neural markers in tissue studies. It is also paired with costimulatory molecules and apoptosis markers in functional assays, and included in large multiplex antibody panels for high-dimensional analysis.

Clone 30-H12 is a widely used monoclonal antibody that specifically recognizes the CD90.2 (Thy-1.2) alloantigen, and scientific literature has revealed several important findings regarding its applications and the biological functions of its target antigen.

Antigen Recognition and Specificity

Clone 30-H12 demonstrates highly specific binding to CD90.2 (Thy-1.2), which is a 28-30 kD GPI-linked membrane glycoprotein belonging to the immunoglobulin superfamily. The antibody shows remarkable specificity, as it does not cross-react with the Thy-1.1 allele found in select mouse strains like AKR/J and PL, nor does it react with rat Thy-1. This specificity is crucial since CD90.1 and CD90.2 differ by only a single amino acid at position 108, where CD90.1 has arginine and CD90.2 has glutamine.

Cellular Distribution and Expression Patterns

Research using clone 30-H12 has mapped the extensive cellular distribution of CD90.2 across multiple tissue types and cell populations. The antibody binds to thymocytes, most peripheral T lymphocytes, some intraepithelial T lymphocytes, epithelial cells, fibroblasts, neurons, and hematopoietic stem cells, but notably not B lymphocytes in most mouse strains. Initially, CD90.2 was reported on thymic dendritic cells, but subsequent investigations revealed that the antigen was likely acquired from T-lineage cells rather than being intrinsically expressed.

Signal Transduction Mechanisms

Studies utilizing clone 30-H12 have uncovered important signal transduction properties of CD90.2. The protein interacts with CD45 in signal transduction pathways and mediates adhesion of thymocytes to thymic stroma. Cross-linking experiments with the 30-H12 antibody have demonstrated that it can induce calcium influx into thymocytes. Furthermore, when co-cross-linked with antibodies to the CD3/TCR complex, it intensifies thymocyte signal transduction, promotes apoptosis of thymocytes, and inhibits the CD3-mediated proliferative response of mature T lymphocytes.

Stem Cell Biology Applications

Clone 30-H12 has proven valuable in stem cell research, where CD90.2 serves as a popular surface marker for various stem cell populations, including hematopoietic stem cells. In flow cytometry applications, it is frequently used in combination with other markers such as CD34 for stem cell identification and characterization. The antibody has also revealed CD90.2's role in inhibiting hematopoietic stem cell proliferation and differentiation.

Immunological Research and Therapeutic Applications

In immunological studies, clone 30-H12 has been employed to investigate T cell responses and vaccine efficacy. Research has used this antibody for T cell depletion studies, administering anti-Thy-1.2 treatments to examine protective immune responses in vaccination models. These studies have contributed to understanding how different T cell populations contribute to immunity against infectious diseases, particularly in the context of tuberculosis research.

Functional Roles in Cell Biology

The use of clone 30-H12 has helped elucidate CD90.2's diverse functional roles beyond immune system applications. Research has shown that CD90.2 binds with ?2 and ?3 integrins and is involved in regulating cell adhesion and neurite outgrowth. This makes it relevant not only for immunological research but also for neurobiology studies examining neuronal development and function.

The extensive citation of clone 30-H12 in scientific literature reflects its reliability and utility as a research tool, contributing significantly to our understanding of CD90.2 biology across multiple biological systems and research applications.