This monoclonal antibody is aseptically packaged and formulated in 0.01 M phosphate buffered saline (150 mM NaCl) PBS pH 7.2 - 7.4 with no carrier protein, potassium, calcium or preservatives added. Due to inherent biochemical properties of antibodies, certain products may be prone to precipitation over time. Precipitation may be removed by aseptic centrifugation and/or filtration.
Product Preparation
Functional grade preclinical antibodies are manufactured in an animal free facility using in vitro cell culture techniques and are purified by a multi-step process including the use of protein A or G to assure extremely low levels of endotoxins, leachable protein A or aggregates.
Storage and Handling
Functional grade preclinical antibodies may be stored sterile as received at 2-8°C for up to one month. For longer term storage, aseptically aliquot in working volumes without diluting and store at ≤ -70°C. Avoid Repeated Freeze Thaw Cycles.
Applications and Recommended Usage? Quality Tested by Leinco
FC The suggested concentration for this PC61 antibody for staining cells in flow cytometry is ≤ 1 μg per 106 cells in a volume of 100 μl. Titration of the reagent is recommended for optimal performance for each application. WB The suggested concentration for this PC61 antibody for use in western blotting is 1-10 μg/ml.
Each investigator should determine their own optimal working dilution for specific applications. See directions on lot specific datasheets, as information may periodically change.
Description
Description
Specificity
Clone PC61 recognizes an epitope on mouse CD25.
Background
CD25, a 55 kD type I transmembrane glycoprotein, has been shown to play roles in lymphocyte differentiation, activation, and proliferation. Many resting memory T cells constitutively express IL2RA. It functions as the receptor for HTLV-1, resulting in its expression on neoplastic cells in adult T cell lymphoma/leukemia. CD25 (sIL-2R) has been used to track disease progression. Some additional clinical applications include Chagas disease, a disease characterized by a decline of CD25 expression on immune cells, and Multiple sclerosis, in which treatments with mAbs target CD25.
Antigen Distribution
CD25 is expressed in activated T cells and B cells, thymocyte subset, pre-B cells, T regulatory cells.
Ligand/Receptor
IL-2
Function
Forms high affinity IL-2R with IL-2Rβ (CD122) and IL-2Rγ (CD132)
Powered by AI: AI is experimental and still learning how to provide the best assistance. It may occasionally generate incorrect or incomplete responses. Please do not rely solely on its recommendations when making purchasing decisions or designing experiments.
In Vivo Applications of Clone PC61 in Mice
Clone PC61 (a monoclonal antibody targeting mouse CD25, the alpha chain of the interleukin-2 receptor) is primarily utilized in vivo for specific immunomodulatory interventions in mouse models.
Depletion of CD25+ Regulatory T Cells (Tregs) The most common application is the selective depletion of CD25+CD4+ Foxp3+ regulatory T cells (Tregs) in vivo. Administering PC61 via intraperitoneal injection has been shown to dramatically reduce (95–98%) CD25+ CD4+ T cells in organs such as the spleen, lungs, and mediastinal lymph nodes (mLNs). This is a well-established method to study the role of Tregs in immune responses, tolerance, autoimmunity, and infection models.
Blockade of IL-2 Signaling Beyond depletion, PC61 can block interleukin-2 (IL-2) binding to its receptor, directly interfering with IL-2-mediated signaling pathways that are critical for T cell proliferation and immune homeostasis. This application is valuable for investigating the contributions of IL-2 signaling to immune cell function and survival in vivo.
Modulation of Immune Responses in Infection Models PC61 has been used to examine the impact of Treg depletion on antiviral immune responses. For example, pre-treatment with PC61 prior to viral challenge (e.g., LCMV infection) alters the dynamics of virus-specific CD8+ T cell responses, though the effects can differ depending on the infection history of the mice (naive vs. pre-immune). In some cases, PC61 treatment has been associated with reduced lung pathology without affecting viral titers, highlighting context-dependent roles of Tregs in immunopathology.
Experimental Protocols
Dosage and Timing: A typical protocol involves a single intraperitoneal injection of 100 μg PC61, administered 3 days prior to the experimental intervention (e.g., infection).
Controls: Isotype control antibodies or PBS are used to ensure specificity of the observed effects.
Validation: Efficacy is confirmed by flow cytometry, showing near-complete depletion of CD25+CD4+ T cells in relevant tissues.
Summary Table
Application
Purpose
Outcome/Notes
Treg depletion
Study immune regulation, tolerance, autoimmunity, infection responses
~95–98% reduction in CD25+CD4+ T cells
IL-2 signaling blockade
Investigate IL-2-dependent immune cell functions
Blocks IL-2 binding, alters T cell responses
Infection model modulation
Assess Treg role in viral immunopathology
Alters CD8+ T cell trafficking and pathology
Key Points
PC61 is a tool for in vivo depletion and functional blockade of CD25+ Tregs in mice.
It is widely used to dissect the roles of Tregs and IL-2 signaling in immune homeostasis and disease models.
Effects are context-dependent, especially in infection models, underscoring the need for appropriate controls.
Clone PC61 remains a cornerstone reagent for in vivo immunological research in mice, enabling precise manipulation of Treg populations and IL-2 signaling to uncover their roles in health and disease.
In the literature, the PC61 antibody is commonly used in conjunction with other antibodies and proteins for various applications, particularly in immunological studies. Here are some of the commonly used antibodies or proteins:
CD4 and Foxp3 Antibodies: These are used alongside PC61 for the identification and functional studies of regulatory T cells (Tregs).
IL-2 (Interleukin-2): PC61 is paired with IL-2 for signaling assays due to its role in blocking IL-2 binding to the CD25 receptor.
Other Anti-CD25 Clones: Clones like 3C7 and 7D4 are used for epitope comparison and studying different aspects of CD25 function.
Additionally, PC61 is often used in flow cytometric analysis, which may involve other antibodies for multi-color staining and identification of different cell populations.
These combinations enhance the understanding of immune cell dynamics, particularly in mouse models where PC61 is extensively used due to its ability to block IL-2 signaling, which is crucial for Treg depletion in various research contexts.
Clone PC61 is a rat monoclonal antibody widely used in mouse models to target CD25 (the interleukin-2 receptor alpha chain) and regulate regulatory T cell (Treg) biology, but its precise mechanism and impact have been clarified in recent literature.
Key findings from PC61 clone citations:
Dual Mechanism: Blockade and Depletion
PC61 can effect both blockade of IL-2 signaling through CD25 and depletion of CD25^+^ Treg cells, but these are distinct outcomes determined by isotype and Fc engineering of the antibody.
Fc-engineered variants of PC61 demonstrated that immune homeostasis can be maintained when only IL-2 signaling is blocked, but active depletion of CD25^+^ Treg cells disrupts homeostasis and leads to immune activation.
Mechanism of Depletion
PC61-mediated depletion relies largely on Fcγ receptor III (FcγRIII)-positive phagocytes, specifically macrophages, not NK cells.
Functional blockade of Fcγ receptors or depletion of phagocytes abolishes the Treg depletion effect of PC61.
Effectiveness and Limitations
In vivo studies often show incomplete depletion (typically 30–70%) of CD25^+^ Treg cells, and residual Treg often display lower or absent CD25 expression.
The rat IgG1 isotype of PC61 has weak effector function in mice; thus, depletion is sometimes inefficient or inconsistent, which has led to misinterpretations in earlier studies regarding its true impact.
Fc-optimized (e.g., murine IgG2a) versions show stronger depletion and improved anti-tumor efficacy—but often require combination therapy (such as with anti-PD1) for significant effects in cancer models.
Functional Blockade Without Depletion
Non-depleting variants of PC61 (e.g., mouse IgG1 or with N297Q mutation) block IL-2-mediated STAT5 phosphorylation but do not lead to Treg cell elimination.
Even with CD25 blockade, some residual CD25 function may persist, helping maintain Treg-mediated immune homeostasis.
Implications
There's now a clear distinction between Treg depletion (affecting both numbers and function and potentially causing immune activation) and pure blockade (mainly affecting IL-2 signaling without massive Treg loss).
These findings highlight the importance of antibody isotype and Fc engineering in the design of anti-CD25 therapeutics and the interpretation of Treg depletion experiments.
Summary Table: Functional Outcomes of PC61 Use
Antibody Variant
Fc Function
Main Effect
Key Cell Type Involved
PC61-rat IgG1
Weak
Partial depletion/blockade
FcγRIII+ macrophages
PC61-murine IgG2a
Strong
Efficient depletion
Macrophages (via FcγRIII)
PC61-mIgG1(N297Q)
None
Blocking only
N/A
Conclusion: The literature consistently shows that PC61's impact varies with its Fc characteristics: it can act as a depleting or blocking antibody, with depletion requiring FcγRIII^+^ phagocytes. This distinction is crucial for experimental interpretation and for the rational design of therapies targeting regulatory T cells.
Dosing regimens of clone PC61 (anti-mouse CD25) in mouse models most commonly use 500 µg per mouse, administered by intraperitoneal injection at weekly intervals, but specific dosing and schedules are often adjusted based on mouse strain, experimental goal, and length of study.
In standard immunology and autoimmunity models (e.g., Treg cell depletion, IL-2 biology, experimental autoimmune encephalomyelitis), PC61 is typically dosed at 500 µg per mouse intraperitoneally, once weekly. This schedule aims to maintain consistent receptor saturation as confirmed by flow cytometry.
The regimen is consistent across both wild-type and genetically modified mice, such as Fcer1 g^−/−^ strains, with this dose and interval providing effective CD25 blockade.
For longer experiments (e.g., 4-week studies), dosing remains at 500 µg per week but may use more animals per group to increase statistical power.
The same dosing strategy is reported in experimental autoimmune encephalomyelitis models, with ongoing clinical monitoring according to the severity of disease progression.
Manufacturers and antibody suppliers also cite 500 µg/mouse/week as the most common regimen for in vivo depletion, but acknowledge that variability exists and regimens can be tailored to mouse model and study design. Alternate schedules, higher frequency, or dose adjustments may occur in models with different pharmacokinetics or when aiming for partial vs. full Treg depletion.
Variability between models:
Some investigators may use higher or lower doses, or more frequent dosing in certain tumor models, particularly where faster or deeper Treg depletion is required, or where the pharmacokinetics of the mouse strain support altered timing.
Antibody isotype variant (e.g., chimeric murine IgG2a vs. rat IgG1) can affect depletion efficacy, especially in tumor models; PC61-mIgG2a variants may be required for improved Treg clearance in certain contexts.
Summary Table: PC61 Dosing in Common Mouse Models
Model/Context
Typical Dose
Route
Frequency
Notes
General immune/Treg depletion
500 µg/mouse
Intraperitoneal
Weekly
For both wild-type and KO mice
Experimental autoimmune dz (EAE)
500 µg/mouse
Intraperitoneal
Weekly
Monitored for clinical signs
Tumor models
500 µg/mouse
Intraperitoneal
Weekly or adjust
Combine with anti-PD-1 for synergy
Alternate regimens
250-500 µg/mouse
Intraperitoneal
3-7 day interval
Tailored to experiment
Key factors influencing regimen selection:
Mouse strain (C57BL/6, BALB/c, KO models)
Experimental duration
Desired degree of Treg depletion
Fc isotype optimization in tumor models for Treg clearance efficacy
The default and most cited regimen remains 500 µg/mouse weekly i.p., but researchers optimize schedules based on their specific experimental models and endpoints.
References & Citations
1.) Braley-Mullen, H. et al. (2018) Immunohorizons. 2(1): 54–66. PubMed
2.) Leonard, WJ. et al. (2002) The EMBO Journal21: 3051
3.) Alt, FW. et al. (1995) Immnnity3: 521
4.) Greene, WC. et al. (1990) J Invest Dermatol.94: 27S
5.) Gubin, M. et al. (2018) Cell.175(4):1014–1030.e19 Journal Link