Anti-Mouse CD90 (Thy-1) (Clone M5/49.4.1) – Purified in vivo GOLD™ Functional Grade

The monoclonal antibody clone M5/49.4.1 is most commonly used in mice for in vivo depletion of CD90 (Thy-1)-positive cells, particularly T cells, and for functionally blocking CD90-mediated processes.

Essential context and supporting details:

• T Cell Depletion: M5/49.4.1 is widely utilized to selectively deplete T cells in mouse models, as CD90 (Thy-1) is a well-established pan-T cell marker in mice. Researchers use this antibody to study the role of T cells in various physiological and disease contexts by observing outcomes after targeted T cell removal.
• Functional Blockade: Beyond depletion, the antibody is also used to block CD90 function in vivo, allowing investigation of CD90's roles in immune responses, fibrosis, and neurodegenerative disease models.
• Application in Disease Models: By depleting or inhibiting CD90+ cells, M5/49.4.1 has been instrumental in dissecting the immune, fibrotic, and neurobiological roles of T cells and other CD90-expressing populations in mice.
• Experimental Confirmation: Studies often administer M5/49.4.1 in vivo via intraperitoneal injection to achieve cell depletion, as confirmed in peer-reviewed research using this clone for depletion of NK and T cells.

Additional relevant uses:

• Flow Cytometry and Immunohistochemistry: M5/49.4.1 may also be employed for identifying and isolating CD90+ cells in mouse tissues, though these are typically ex vivo applications.

In summary, depletion of T cells in vivo is the hallmark application of clone M5/49.4.1 in mice, with secondary uses in blocking and analyzing the functional properties of CD90+ cell populations in immunological and other biological studies.

Commonly, M5/49.4.1 (anti-mouse CD90/Thy-1) is used in combination with antibodies that target other cell surface markers in mouse immunology and depletion studies. The most frequently co-used antibodies or proteins include:

• Anti-CD3 antibodies: CD3 is a pan-T cell marker and anti-CD3 antibodies (such as clone 145-2C11) are often used for T cell identification, depletion, or activation, together with anti-CD90.
• Anti-CD4 and anti-CD8 antibodies: These are used to distinguish T cell subsets (helper vs. cytotoxic T cells) in the same experimental setups as CD90, helping to delineate specific T cell populations.
• Anti-CD45: This is a pan-leukocyte marker and is sometimes used either as a control or to further refine gating in flow cytometry experiments.
• Lineage marker antibodies (such as anti-CD11b or anti-F4/80): These may be used alongside anti-CD90 to discriminate T cells from myeloid or other lineages, especially in mouse immune profiling studies.
• Isotype controls: Control antibodies of the same isotype as M5/49.4.1, but lacking specificity (such as Rat IgG2a isotype controls), are routinely co-used.
• Anti-BMAL1, anti-beta-actin, anti-PGP: In multicolor panels or Western blots, additional antibodies such as anti-BMAL1 (transcription factor), anti-beta-actin (loading control), and anti-PGP (permeability glycoprotein) may be included depending on the specific experimental goals (as reported in blood-brain barrier studies where M5/49.4.1 was used for cell selection).

These combinations allow researchers to:

• Deplete specific T cell subsets in vivo.
• Phenotype cell populations by flow cytometry.
• Control for non-specific binding and background signal.

The choice of which antibodies to use alongside M5/49.4.1 depends on the tissue, the cell types of interest, and the broader immunophenotyping or functional analysis required.

Clone M5/49.4.1 is a rat monoclonal antibody (IgG2a, κ) targeting mouse Thy-1 (CD90), a highly conserved glycoprotein that has been extensively used in immunological research. The antibody was originally generated by immunizing (Lewis x BN) F1 rats with a secondary mixed lymphocyte culture consisting of C57BL/6 mouse anti-irradiated BN rat lymphoma, with spleen cells subsequently fused with NS-1 myeloma cells to create hybridomas that bind cytolytic T lymphocyte surface molecules.

Target Protein Characteristics

Thy-1/CD90 is a 25-35 kDa GPI-anchored protein belonging to the immunoglobulin superfamily. The molecule is heavily N-glycosylated, with carbohydrate content comprising up to 40% of its molecular mass, and its moiety composition varies between tissues as well as between cells of the same lineage at different developmental stages. In mice, Thy-1 exists in two allelic forms: Thy1.1 (found in AKR/J and PL strains) and Thy1.2 (expressed by most mouse strains), distinguished by a single amino acid difference at position 89.

Expression Patterns and Cellular Distribution

M5/49.4.1 recognizes CD90 expressed by thymocytes, peripheral T cells, myoblasts, epidermal cells, and keratinocytes in mice. The antibody is considered a pan T cell marker in mice, making it particularly valuable for identifying and studying various T cell populations. The molecule has been shown to interact with CD45 in signal transduction pathways during lymphocyte proliferation.

Functional Roles and Applications

Research utilizing this clone has revealed that while Thy-1 deficiency does not completely compromise immunity, the protein's presence or absence modulates phenotypes in certain cancers, fibrotic diseases, and neuronal injury. The effects of Thy-1 are notably context-dependent, with the molecule playing roles in regulating cell adhesion, apoptosis, metastasis, inflammation, and fibrosis.

The M5/49.4.1 antibody has proven particularly useful for T lymphocyte depletion studies. Scientific literature demonstrates its application in functional assays, including the depletion of NKR+ cells through injections of 250 µg anti-Thy1, and in cell isolation procedures where it has been used for labeling cells prior to magnetic bead separation.

Research Applications

The clone is widely utilized across multiple experimental techniques, including flow cytometry, functional assays, immunoprecipitation, and depletion studies. For flow cytometry applications, the recommended concentration is 2 μg/mL for 1×10⁵-5×10⁵ cells, with verified performance on C57/BL6 mouse splenocytes. The antibody's versatility extends to in vivo studies, where it serves as a functional-grade reagent for investigating immune cell populations and their roles in various biological processes.

Dosing regimens for the M5/49.4.1 antibody clone can vary considerably across different mouse models and experimental applications, with investigators needing to determine optimal protocols for their specific research contexts.

Application-Specific Dosing

The antibody is used across several experimental applications including depletion studies, flow cytometry (FA), and immunoprecipitation (IP), each requiring different dosing approaches. Each investigator should determine their own optimal working dilution for specific applications, as information may periodically change based on lot-specific requirements.

Documented Dosing Examples

In practical research settings, specific dosing regimens have been documented. For antigen presentation assays, splenic APCs are depleted of T cells using anti-Thy1 antibody at 50 μg/ml (clone M5/49.4.1), combined with 1:20 rabbit complement, incubated for 1 hour at 37°C. This same concentration (50 μg/ml) has been used in other experimental protocols involving T cell studies.

For in vivo studies, the dosing can vary significantly based on the experimental endpoint. When used in adoptive transfer models, such as diabetes induction studies in Rag1−/− NOD recipients, the antibody protocol is part of a broader experimental design where recipient mice receive daily injections of test compounds while the M5/49.4.1 antibody is used for initial T cell depletion from APCs.

Model-Dependent Considerations

The variation in dosing regimens stems from differences in mouse strain characteristics, experimental objectives, and the specific immune cell populations being targeted. Since CD90 (Thy-1) expression varies between different cell types—including thymocytes, peripheral T-cells, myoblasts, epidermal cells, and keratinocytes—the antibody concentration must be adjusted accordingly. Additionally, mice express two different Thy1 alleles (Thy1.1 in AKR/J and PL strains, and Thy1.2 in most other strains), which may influence antibody binding efficiency and required dosing.