Anti-Mouse CD25 Monoclonal Antibodies for Flow Cytometry

PA007489.r1: Recombinant Anti-mouse CD25 Monoclonal Antibody (Clone: PC61.5.3), Rat IgG1 Lambda

Recombinant Rat IgG1 Monoclonal Antibody. 
Clone: PC61.5.3.
Isotype:  Rat IgG1 Lambda.
Source: The anti-mouse CD25 monoclonal antibody (clone: PC61.5.3) was produced in mammalian cells.
Conjugation: Non-conjugated.
Specificity/Sensitivity: The recombinant PC61.5.3 antibody specifically binds to mouse CD25 protein.
Applications: Flow cytometry (FC), and Immunohistochemistry - Frozen (IHC-F). 
Form of Antibody: 0.2 uM filtered solution, pH 7.4, containing 0.09% sodium azide.
Purity: >95% by SDS-PAGE under reducing conditions.

PA007489.h1Fs: Recombinant Anti-mouse CD25 Monoclonal Antibody (Clone: PC61.5.3), Human IgG1 Fc Silent Kappa

Recombinant Human IgG1 Monoclonal Antibody with Fc silent mutation. 
Clone: PC61.5.3.
Isotype: Human IgG1 kappa.
Source: The anti-mouse CD25 monoclonal antibody (clone: PC61.5.3) was produced in mammalian cells.
Conjugation: Non-conjugated.
Specificity/Sensitivity: The recombinant PC61.5.3 antibody specifically binds to mouse CD25 protein.
Applications: Flow cytometry (FC), and Immunohistochemistry - Frozen (IHC-F). 
Form of Antibody: 0.2 uM filtered solution, pH 7.4, containing 0.09% sodium azide.
Purity: >95% by SDS-PAGE under reducing conditions.

Shipping: The PC61.5.3 antibody is shipped with ice pack. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles. 
12 months from date of receipt, 2 to 8°C as supplied. Do not freeze it.

Background
The in vivo grade recombinant anti-mouse CD25 monoclonal antibodies are based on the rat monoclonal antibody anti-CD25 (mouse) with the clone number of PC61, PC61.5 or PC61.5.3. The rat IgG1 lamda monoclonal antibody has been validated and reported for use in immunoprecipitation (IP), immunohistochemistry (IHC), Flow Cytometry (FC), and various functional assays, such as in vivo depletion of CD25+CD4+ Treg cells, in vitro blocking of IL-2 binding to low- and high-affinity receptors, and growth inhibition of IL-2-dependent T-cell lines. Expression of CD25, together with CD4 and FOXP3, is considered a phenotypic signature for Treg cells. In vivo depletion of mouse CD25(+)CD4(+)FoxP3(+) Treg cells using the rat PC61 monoclonal antibody is widely used to characterize the Treg function. The PC61 monoclonal antibody specifically binds to an epitope of CD25 which is distinct from the IL-2 binding site and from those recognized by the 3C7 and 7D4 monoclonal antibodies. By doing so, the antibody blocks binding of IL-2 to CD25.

References of anti-mouse CD25 antibody for Flow Cytometry (Clone: PC61.5.3):

Protocol to examine immune subpopulations in murine conjunctiva and lacrimal gland using flow cytometry
Ma, B., Zhou, Y., Duan, H., Sun, Z., Han, W., Qi, H. STAR Protocols. 2024 Mar 15;5(1):102892. doi: 10.1016/j.xpro.2024.102892. PMID: 38458908
Add 100 μL of antibody cocktail (containing anti-CD45, anti-CD4, anti-CD8, anti-NK1.1, anti-γδTCR, anti-CD11b, anti-F4/80, anti-Ly6C, anti-Ly6G, anti-CD19, anti-CD25 (clone: PC61.5), and anti-Foxp3 antibodies) to each tube and incubate at 4°C for 30 min in the dark. Resuspend cells in 200 μL of FACS buffer and transfer to a 96-well U-bottom plate for flow cytometry analysis. After centrifugation, resuspend cells in 100 μL of fixation/permeabilization solution and incubate at 4°C for 30 min in the dark for intracellular staining. Analyze samples using a flow cytometer (e.g., BD LSRFortessa or equivalent) and process data with FlowJo software. Ensure single-stain controls and fluorescence minus one (FMO) controls are included for compensation and gating accuracy.
Tags: activity of PC61.5.3 antibody; activity of PC61.5.3 mAb

Do more with less: improving high parameter cytometry through overnight staining
Whyte, C. E., Tumes, D. J., Liston, A., Burton, O. T. Current Protocols. 2022 Nov;2(11):e589. doi: 10.1002/cpz1.589. PMID: 36315106
For Treg panel staining: CD45 (30-F11), CD3 (145-2C11), CD4 (RM4-5), CD25 (PC61.5), Foxp3 (FJK-16s), Helios (22F6), Neuropilin-1 (3E12), CD44 (IM7), CD62L (MEL-14). Resuspend cells in 100 μL of the antibody cocktail diluted in FACS buffer and incubate for 30 min at 4°C in the dark. After surface staining, fix and permeabilize cells using the Foxp3/Transcription Factor Staining Buffer Set for 30 min at 4°C. Analyze samples on a flow cytometer such as the BD LSRFortessa or Cytek Aurora, ensuring proper compensation controls are included. Overnight staining at 4°C can improve signal intensity for certain fluorochromes without compromising cell viability or data quality.
Tags: function of PC61.5.3 mAb; PC61.5 antibody

Partial CD25 Antagonism Enables Dominance of Antigen-Inducible CD25high FOXP3+ Regulatory T Cells As a Basis for a Regulatory T Cell-Based Adoptive Immunotherapy
Wilkinson, D. S., Ghosh, D., Nickle, R. A., Moorman, C. D., Mannie, M. D. Frontiers in Immunology. 2017 Dec 14;8:1782. doi: 10.3389/fimmu.2017.01782. PMID: 29308488
The PC61.5 antibody, a rat IgG1 anti-mouse CD25 mAb, was purified from hybridoma supernatants (PC61 5.3; ATCC) by protein G affinity chromatography. Splenocytes were stained with fluorescently conjugated antibodies against CD4, CD25, and Foxp3 to assess Treg populations by flow cytometry. Partial CD25 antagonism with PC61.5 enabled selective depletion of low-affinity Tregs while preserving antigen-specific CD25high Foxp3+ Tregs. Flow cytometry was performed using a BD FACSCanto II, and data were analyzed with FlowJo software to quantify Treg subpopulations. Mice were injected intraperitoneally with 200 μg of PC61.5 antibody to modulate Treg activity in vivo.
Tags: PC61.5 antibody for flow cytometry; PC61.5 antibody for mouse tumor model

For more references about anti-mouse CD25 antibody for Flow Cytometry (Clone: PC61.5.3), please contact our scientific support team with message@sydlabs.com.

Syd Labs provides the following in vivo grade recombinant anti-mouse CD25 monoclonal antibodies:
Recombinant Anti-mouse CD25 monoclonal antibodies (Clone: PC61.5.3)

Syd Labs also provides the following research grade antibody biosimilar proteins:
Daclizumab Biosimilar, research grade, Anti-human CD25 Monoclonal Antibody