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PerCP-Cy™5.5 Rat Anti-Mouse CD25
PerCP-Cy™5.5 Rat Anti-Mouse CD25
Flow cytometric analysis of CD25 expression on stimulated mouse splenocytes. Freshly prepared mouse splenic leucocytes were stimulated with concanavalin A for 3 days, then stained with either PerCP-Cy5.5 Rat IgG1, λ Isotype Control (Cat. No. 551072; dashed line histogram) or PerCP-Cy5.5 Rat Anti-Mouse CD25 antibody (Cat. No. 551071/ 561112; solid line histogram) at 1 µg/test. Fluorescence histogram showing CD25 (or Ig Isotype) expression was derived from gated events with the forward and side light- scatter characteristics of intact leucocytes. Flow cytometry and data analysis were performed using a BD FACSCanto™ Cell Analyzer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.
Flow cytometric analysis of CD25 expression on stimulated mouse splenocytes. Freshly prepared mouse splenic leucocytes were stimulated with concanavalin A for 3 days, then stained with either PerCP-Cy5.5 Rat IgG1, λ Isotype Control (Cat. No. 551072; dashed line histogram) or PerCP-Cy5.5 Rat Anti-Mouse CD25 antibody (Cat. No. 551071/ 561112; solid line histogram) at 1 µg/test. Fluorescence histogram showing CD25 (or Ig Isotype) expression was derived from gated events with the forward and side light- scatter characteristics of intact leucocytes. Flow cytometry and data analysis were performed using a BD FACSCanto™ Cell Analyzer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.
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_394031
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 PerCP-Cy5.5 under optimum conditions, and unconjugated antibody and free PerCP-Cy5.5 were removed. Storage of PerCP-Cy5.5 conjugates in unoptimized diluent is not recommended and may result in loss of signal intensity.

Recommended Assay Procedures

For detection of low-density CD25 expression, we recommend the use of the PE conjugate of PC61 antibody (Cat. No. 553866) or the biotin conjugate of the 7D4 antibody (Cat. No. 553069/553070) with a "bright" second-step reagent, such as Streptavidin-PE (Cat. No. 554061). For tandem conjugates incorporating PerCP (e.g., PerCP-Cy5.5), the excitation and emission properties of PerCP and the kinetics of energy exchange between the fluorochromes of the tandem dye may limit their effectiveness on high-speed and/or sorting flow cytometers. Therefore, for third-color flow-cytometric analysis using ≥25-mW laser power, we recommend PE-Cy7-conjugated reagents (e.g., Cat. No. 552880).

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.

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. 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.
  3. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  4. PerCP-Cy5.5–labelled antibodies can be used with FITC- and R-PE–labelled reagents in single-laser flow cytometers with no significant spectral overlap of PerCP-Cy5.5, FITC, and R-PE fluorescence.
  5. PerCP-Cy5.5 is optimized for use with a single argon ion laser emitting 488-nm light. Because of the broad absorption spectrum of the tandem fluorochrome, extra care must be taken when using dual-laser cytometers, which may directly excite both PerCP and Cy5.5™. 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. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  8. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  9. Cy is a trademark of Global Life Sciences Solutions Germany GmbH or an affiliate doing business as Cytiva.
551071 Rev. 11
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.

551071 Rev. 11
Format Details
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PerCP-Cy5.5
PerCP-Cy5.5 dye is part of the BD blue family of dyes. This tandem fluorochrome is comprised of a fluorescent protein complex (PerCP) with an excitation maximum (Ex Max) of 482 nm and an acceptor dye with an emission maximum (Em Max) at 676 nm. PerCP-Cy5 is designed to be excited by the blue laser (488-nm) and detected using an optical filter centered near 680 nm (e.g., a 695/40 nm bandpass filter). The donor dye can be partially excited by the Violet (405-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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PerCP-Cy5.5
Blue 488 nm
482 nm
676 nm
551071 Rev.11
Citations & References
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Development References (15)

  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. Crowley M, Inaba K, Witmer-Pack M, Steinman RM. The cell surface of mouse dendritic cells: FACS analyses of dendritic cells from different tissues including thymus. Cell Immunol. 1989; 118(1):108-125. (Biology). View Reference
  4. 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
  5. Godfrey DI, Zlotnik A. Control points in early T-cell development. Immunol Today. 1993; 14(11):547-553. (Biology). View Reference
  6. Greimers R, Trebak M, Moutschen M, Jacobs N, Boniver J. Improved four-color flow cytometry method using fluo-3 and triple immunofluorescence for analysis of intracellular calcium ion ([Ca2+]i) fluxes among mouse lymph node B- and T-lymphocyte subsets. Cytometry. 1996; 23(3):205-217. (Biology). View Reference
  7. 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). View Reference
  8. 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
  9. 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
  10. Pollard AM, Lipscomb MF. Characterization of murine lung dendritic cells: similarities to Langerhans cells and thymic dendritic cells. J Exp Med. 1990; 172(1):159-167. (Biology). View Reference
  11. 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
  12. Rolink A, Grawunder U, Winkler TH, Karasuyama H, Melchers F. IL-2 receptor alpha chain (CD25, TAC) expression defines a crucial stage in pre-B cell development. Int Immunol. 1994; 6(8):1257-1264. (Biology). View Reference
  13. Shapiro HM. Practical Flow Cytometry, 3rd Edition. New York: Wiley-Liss, Inc; 1995:280-281.
  14. Takahashi T, Tagami T, Yamazaki S, et al. Immunologic self-tolerance maintained by CD25(+)CD4(+) regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J Exp Med. 2000; 192(2):303-309. (Biology). View Reference
  15. Taniguchi T, Minami Y. The IL-2/IL-2 receptor system: a current overview. Cell. 1993; 73(1):5-8. (Biology). View Reference
View All (15) View Less
551071 Rev. 11

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