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PE-Cy™7 Rat Anti-Mouse CD25
PE-Cy™7 Rat Anti-Mouse CD25

Two-color analysis of the expression of CD25 in bone marrow. BALB/c bone marrow leukocytes were stained with FITC Rat Anti-Mouse CD45R/B220 (Cat. No. 553087/553088) and either PE-Cy™7 Rat IgG1, λ Isotype Control (Cat. No. 552869; left panel) or PE-Cy™7 Rat Anti-Mouse CD25 (Cat. No. 552880/561780; right panel). Please note that the dead leukocytes were not excluded in this experiment, and the typical diagonal dead-cell population appears in the left panel. The same dead-cell population is partially obscured in the right panel. Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

Two-color analysis of the expression of CD25 in bone marrow. BALB/c bone marrow leukocytes were stained with FITC Rat Anti-Mouse CD45R/B220 (Cat. No. 553087/553088) and either PE-Cy™7 Rat IgG1, λ Isotype Control (Cat. No. 552869; left panel) or PE-Cy™7 Rat Anti-Mouse CD25 (Cat. No. 552880/561780; right panel). Please note that the dead leukocytes were not excluded in this experiment, and the typical diagonal dead-cell population appears in the left panel. The same dead-cell population is partially obscured in the right panel. Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

Product Details
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BD Pharmingen™
Interleukin-2 receptor alpha chain; IL-2RA; IL-2Rα; Il2ra; IL-2R p55
Mouse (QC Testing)
Rat OFA, also known as Outbred OFA IgG1, λ
IL-2-dependent cytolytic mouse T-cell clone B6.1
Flow cytometry (Routinely Tested)
0.2 mg/ml
16184
AB_394509
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with PE-Cy7 under optimum conditions, and unconjugated antibody and free PE-Cy7 were removed.

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. An isotype control should be used at the same concentration as the antibody of interest.
  3. 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.
  4. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  5. PE-Cy7 is a tandem fluorochrome composed of R-phycoerythrin (PE), which is excited by 488-nm light and serves as an energy donor, coupled to the cyanine dye Cy7, which acts as an energy acceptor and fluoresces maximally at 780 nm. PE-Cy7 tandem fluorochrome emission is collected in a detector for fluorescence wavelengths of 750 nm and higher. Although every effort is made to minimize the lot-to-lot variation in the efficiency of the fluorochrome energy transfer, differences in the residual emission from PE may be observed. Therefore, we recommend that individual compensation controls be performed for every PE-Cy7 conjugate. PE-Cy7 is optimized for use with a single argon ion laser emitting 488-nm light, and there is no significant overlap between PE-Cy7 and FITC emission spectra. When using dual-laser cytometers, which may directly excite both PE and Cy7, we recommend the use of cross-beam compensation during data acquisition or software compensation during data analysis.
  6. Please observe the following precautions: Absorption of visible light can significantly alter the energy transfer occurring in any tandem fluorochrome conjugate; therefore, we recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to prevent exposure of conjugated reagents, including cells stained with those reagents, to room illumination.
  7. Warning: Some APC-Cy7 and PE-Cy7 conjugates show changes in their emission spectrum with prolonged exposure to formaldehyde. If you are unable to analyze fixed samples within four hours, we recommend that you use BD™ Stabilizing Fixative (Cat. No. 338036).
  8. Cy is a trademark of GE Healthcare.
  9. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
552880 Rev. 5
Antibody Details
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PC61

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.

552880 Rev. 5
Format Details
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PE-Cy7
PE-Cy7 dye is a part of the BD PE family of dyes. This tandem fluorochrome is comprised of a R-Phycoerythrin (PE) donor that has excitation maxima (Ex Max) of 496-nm and 566-nm and an acceptor dye, Cy™7, with an emission maximum (Em Max) at 781-nm. PE can be excited by the Blue (488-nm), Green (532-nm) and yellow-green (561-nm) lasers and detected using an optical filter centered near 781 nm (e.g., a 760/60-nm bandpass filter). The donor dye can be excited by the Blue (488-nm), Green (532-nm) and yellow-green (561-nm) lasers and the acceptor dye can be excited by the Red (627–640-nm) laser resulting in cross-laser excitation and fluorescence spillover. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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PE-Cy7
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
781 nm
552880 Rev.5
Citations & References
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Development References (8)

  1. 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
  2. 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
  3. 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
  4. Godfrey DI, Zlotnik A. Control points in early T-cell development. Immunol Today. 1993; 14(11):547-553. (Biology). View Reference
  5. 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: Blocking, Flow cytometry). View Reference
  6. 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). View Reference
  7. 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
  8. 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
View All (8) View Less
552880 Rev. 5

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For Research Use Only. Not for use in diagnostic or therapeutic procedures.