BD Horizon™ BUV737 Rat Anti-Mouse CD25 (IL-2 Receptor α)
Clone PC61 (RUO)
Flow cytometric analysis of CD25 (IL-2 Receptor α) expression on unstimulated and stimulated Mouse splenic leukocytes.
Left and Middle Plots: Mouse splenic leukocytes were preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) [Cat. No. 553142]. The cells were then stained with PE Rat Anti-Mouse CD4 antibody (Cat. No. 553049) and with either BD Horizon™ BUV737 Rat IgG1, κ Isotype Control (Cat. No. 612770; Left Plot) or BD Horizon™ BUV737 Rat Anti-Mouse CD25 (IL-2 Receptor α) antibody (Cat. No. 569600/569601; Middle Plot) at 0.5 µg/test. DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) Solution (Cat. No. 564907) was added to cells right before analysis. The bivariate pseudocolor density plot showing the correlated expression of CD25 (IL-2 Receptor α) [or Ig Isotype control staining] versus CD4 was derived from DAPI negative-gated events with the forward and side light-scatter characteristics of viable leukocytes.
Right Plot: Mouse splenic leukocytes were stimulated for 3 days with Concanavalin A (ConA). The cells were preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody and then stained with either BD Horizon™ BUV737 Rat IgG1, κ Isotype Control (dashed line histogram) or BD Horizon™ BUV737 Rat Anti-Mouse CD25 (IL-2 Receptor α) antibody (solid line histogram) at 0.5 µg/test. The fluorescence histogram showing CD25 (IL-2 Receptor α) expression (or Ig Isotype control staining) was derived from DAPI negative-events with the forward and side light-scatter characteristics of viable lymphoblasts.
Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Flow Cytometer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.
Flow cytometric analysis of CD25 (IL-2 Receptor α) expression on unstimulated and stimulated Mouse splenic leukocytes.
Left and Middle Plots: Mouse splenic leukocytes were preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) [Cat. No. 553142]. The cells were then stained with PE Rat Anti-Mouse CD4 antibody (Cat. No. 553049) and with either BD Horizon™ BUV737 Rat IgG1, κ Isotype Control (Cat. No. 612770; Left Plot) or BD Horizon™ BUV737 Rat Anti-Mouse CD25 (IL-2 Receptor α) antibody (Cat. No. 569600/569601; Middle Plot) at 0.5 µg/test. DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) Solution (Cat. No. 564907) was added to cells right before analysis. The bivariate pseudocolor density plot showing the correlated expression of CD25 (IL-2 Receptor α) [or Ig Isotype control staining] versus CD4 was derived from DAPI negative-gated events with the forward and side light-scatter characteristics of viable leukocytes.
Right Plot: Mouse splenic leukocytes were stimulated for 3 days with Concanavalin A (ConA). The cells were preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody and then stained with either BD Horizon™ BUV737 Rat IgG1, κ Isotype Control (dashed line histogram) or BD Horizon™ BUV737 Rat Anti-Mouse CD25 (IL-2 Receptor α) antibody (solid line histogram) at 0.5 µg/test. The fluorescence histogram showing CD25 (IL-2 Receptor α) expression (or Ig Isotype control staining) was derived from DAPI negative-events with the forward and side light-scatter characteristics of viable lymphoblasts.
Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Flow Cytometer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.
Preparation And Storage
Recommended Assay Procedures
BD® CompBeads can be used as surrogates to assess fluorescence spillover (compensation). When fluorochrome conjugated antibodies are bound to BD® CompBeads, they have spectral properties very similar to cells. However, for some fluorochromes there can be small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls. It is strongly recommended that when using a reagent for the first time, users compare the spillover on cells and BD® CompBeads to ensure that BD® CompBeads are appropriate for your specific cellular application.
For optimal and reproducible results, BD Horizon Brilliant™ Stain Buffer should be used anytime BD Horizon Brilliant dyes are used in a multicolor flow cytometry panel. Fluorescent dye interactions may cause staining artifacts which may affect data interpretation. The BD Horizon Brilliant Stain Buffer was designed to minimize these interactions. When BD Horizon Brilliant Stain Buffer is used in the multicolor panel, it should also be used in the corresponding compensation controls for all dyes to achieve the most accurate compensation. For the most accurate compensation, compensation controls created with either cells or beads should be exposed to BD Horizon Brilliant Stain Buffer for the same length of time as the corresponding multicolor panel. More information can be found in the Technical Data Sheet of the BD Horizon Brilliant Stain Buffer (Cat. No. 563794/566349) or the BD Horizon Brilliant Stain Buffer Plus (Cat. No. 566385).
Note: When using high concentrations of antibody, background binding of this dye to erythroid cell subsets (mature erythrocytes and precursors) has been observed. For researchers studying these cell populations, or in cases where light scatter gating does not adequately exclude these Cells from the analysis, this background may be an important factor to consider when selecting reagents for panel(s).
Product Notices
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- An isotype control should be used at the same concentration as the antibody of interest.
- BD Horizon Brilliant Ultraviolet 737 is covered by one or more of the following US patents: 8,110,673; 8,158,444; 8,227,187; 8,575,303; 8,354,239.
- BD Horizon Brilliant Stain Buffer is covered by one or more of the following US patents: 8,110,673; 8,158,444; 8,575,303; 8,354,239.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- For U.S. patents that may apply, see bd.com/patents.
Companion Products
The PC61 monoclonal antibody specifically binds to CD25, the low-affinity IL-2 Receptor α chain (IL-2Rα, p55) expressed on activated T and B lymphocytes from all mouse strains tested. IL-2Rα by itself is not a signaling receptor. However, it can combine with IL-2 Receptor β (CD122) and γc (CD132) chains to form high-affinity, signaling receptor complexes for IL-2. Resting T and B lymphocytes and resting and activated NK cells do not express IL-2Rα. CD25 is transiently expressed at a low level during normal B-cell development in the bone marrow on the CD45R/B220low TdT- sIg- Pre-B/Pre-B-II and CD45R/B220low TdT- sIgM+ sIgD- immature B stages, but not on the CD45R/B220low TdT+ sIg- Pro-B/Pre-B-I stage nor on CD45R/B220high TdT- sIgM+ sIgD+ mature B cells. It is expressed at a higher level during a very early stage of T-cell development in fetal and adult thymus. Peripheral CD25+CD4+ lymphocytes called regulatory T (Treg) cells are involved in the maintenance of self-tolerance. It has also been reported that dendritic cells express CD25, recognized by mAb 7D4. The PC61 antibody recognizes an epitope of CD25 which is distinct from the IL-2 binding site and from those recognized by mAbs 3C7 and 7D4. It blocks binding of IL-2 to CD25, presumably by inducing a conformational change in CD25.
Development References (9)
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Ceredig R, Lowenthal JW, Nabholz M, MacDonald HR. Expression of interleukin-2 receptors as a differentiation marker on intrathymic stem cells. Nature. 1985; 314(6006):98-100. (Clone-specific: Blocking, Immunohistochemistry). View Reference
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Chen J, Ma A, Young F, Alt FW. IL-2 receptor alpha chain expression during early B lymphocyte differentiation. Int Immunol. 1994; 6(8):1265-1268. (Biology). View Reference
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Ernst DN, Weigle WO, McQuitty DN, Rothermel AL, Hobbs MV. Stimulation of murine T cell subsets with anti-CD3 antibody. Age-related defects in the expression of early activation molecules. J Immunol. 1989; 142(5):1413-1421. (Clone-specific: Flow cytometry). View Reference
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Garni-Wagner BA, Witte PL, Tutt MM, et al. Natural killer cells in the thymus. Studies in mice with severe combined immune deficiency. J Immunol. 1990; 144(3):796-803. (Biology). View Reference
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Godfrey DI, Zlotnik A. Control points in early T-cell development. Immunol Today. 1993; 14(11):547-553. (Biology). View Reference
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Lowenthal JW, Corthésy P, Tougne C, Lees R, MacDonald HR, Nabholz M. High and low affinity IL 2 receptors: analysis by IL 2 dissociation rate and reactivity with monoclonal anti-receptor antibody PC61. J Immunol. 1985; 135(6):3988-3994. (Immunogen: Bioassay, Blocking, Functional assay, Inhibition, Radioimmunoassay). View Reference
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Lowenthal JW, Zubler RH, Nabholz M, MacDonald HR. Similarities between interleukin-2 receptor number and affinity on activated B and T lymphocytes. Nature. 1985; 315(6021):669-672. (Clone-specific: Blocking, Immunoprecipitation, Radioimmunoassay). View Reference
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Moreau JL, Nabholz M, Diamantstein T, Malek T, Shevach E, Theze J. Monoclonal antibodies identify three epitope clusters on the mouse p55 subunit of the interleukin 2 receptor: relationship to the interleukin 2-binding site. Eur J Immunol. 1987; 17(7):929-935. (Clone-specific: Blocking). View Reference
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Read S, Malmstrom V, Powrie F. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25(+)CD4(+) regulatory cells that control intestinal inflammation. J Exp Med. 2000; 192(2):295-302. (Biology). View Reference
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.