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Mouse B Lymphocyte Activation Antibody Cocktail, with Isotype Control; PE-Cy™7 CD25, PE CD69, & APC CD19

Mouse B Lymphocyte Activation Antibody Cocktail, with Isotype Control; PE-Cy™7 CD25, PE CD69, & APC CD19

(RUO)
Mouse B Lymphocyte Activation Antibody Cocktail, with Isotype Control; PE-Cy™7 CD25, PE CD69, & APC CD19

Identification of activated B lymphocytes using Mouse B Lymphocyte Activation Antibody Cocktail, with Isotype Control. BALB/c splenocytes were activated by culture for 48 hours with anti-IgM antibody (Jackson immunoresearch) and stained with either Mouse B Lymphocyte Activation Isotype Control (left panels) or Mouse B Lymphocyte Activation Antibody Cocktail (middle panels). Unactivated BALB/c splenocytes were stained with Mouse B Lymphocyte Activation Antibody Cocktail (right panels) or Mouse B Lymphocyte Activation Isotype Control (not shown). Scatter plots were used to select either activated lymphoblasts (left and middle panels) or resting lymphocytes (right panels) for data analysis. The two-color contour plots display the CD19+ B lymphocytes which express the activation antigens CD25 (top of middle and right panels) and CD69 (bottom of middle and right panels). Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

Identification of activated B lymphocytes using Mouse B Lymphocyte Activation Antibody Cocktail, with Isotype Control. BALB/c splenocytes were activated by culture for 48 hours with anti-IgM antibody (Jackson immunoresearch) and stained with either Mouse B Lymphocyte Activation Isotype Control (left panels) or Mouse B Lymphocyte Activation Antibody Cocktail (middle panels). Unactivated BALB/c splenocytes were stained with Mouse B Lymphocyte Activation Antibody Cocktail (right panels) or Mouse B Lymphocyte Activation Isotype Control (not shown). Scatter plots were used to select either activated lymphoblasts (left and middle panels) or resting lymphocytes (right panels) for data analysis. The two-color contour plots display the CD19+ B lymphocytes which express the activation antigens CD25 (top of middle and right panels) and CD69 (bottom of middle and right panels). Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

Product Details
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BD Pharmingen™
Mouse (QC Testing)
Flow cytometry (Routinely Tested)
RUO
AB_2275535


Description

The Mouse B Lymphocyte Activation Antibody Cocktail is a three-color reagent designed to identify major subsets of B lymphocytes by direct immunofluorescent staining with flow cytometric analysis. The PC61 antibody reacts with CD25, the low-affinity IL-2 Receptor achain (IL-2Rα, p55) expressed on activated T and B lymphocytes from all mouse strains tested. CD25 is also found on some developing B cells in the bone marrow, early developing T cells in the thymus, peripheral CD4+ regulatory T (Treg) cells, and dendritic cells. The H1.2F3 antibody reacts with CD69 (Very Early Activation antigen). Its expression is rapidly induced upon activation of lymphocytes (T, B, NK, and NK-T cells) neutrophils, and macrophages. CD69 is also expressed on thymocytes that are undergoing positive selection. The 1D3 antibody reacts with CD19, a B lymphocyte-lineage differentiation antigen that is expressed throughout B-lymphocyte development from the pro-B cell through the mature B-cell stages. Terminally differentiated plasma cells do not express CD19. The three antibodies have been titrated and pre-diluted, mixed together, and formulated for optimal staining performance. The Mouse B Lymphocyte Activation Isotype Control contains equivalent concentrations of fluorochrome- and isotype-matched negative-control immunoglobulin.

The use of three different fluorochromes for the labeling of the three different antibodies permits the recognition of each of the three antigens on each cell in a sample. The expression of the three antigens identifies activated B lymphocytes. Additional fluorochrome-labeled reagents may be combined with the Mouse B Lymphocyte Activation Antibody Cocktail, and the Mouse B Lymphocyte Activation Isotype Control, to further characterize activated B-cell subpopulations.

Preparation And Storage

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. The antibody was conjugated with R-PE under optimum conditions, and unconjugated antibody and free PE were removed. The antibody was conjugated to APC under optimum conditions, and unconjugated antibody and free APC were removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.

Product Notices

  1. This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
  2. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  3. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  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. This APC-conjugated reagent can be used in any flow cytometer equipped with a dye, HeNe, or red diode laser.
  8. 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).
  9. Cy is a trademark of Amersham Biosciences Limited. This conjugated product is sold under license to the following patents: US Patent Nos. 5,486,616; 5,569,587; 5,569,766; 5,627,027.
  10. This product is subject to proprietary rights of Amersham Biosciences Corp. and Carnegie Mellon University and made and sold under license from Amersham Biosciences Corp. This product is licensed for sale only for research. It is not licensed for any other use. If you require a commercial license to use this product and do not have one return this material, unopened to BD Biosciences, 10975 Torreyana Rd, San Diego, CA 92121 and any money paid for the material will be refunded.
  11. 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.
  12. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
558063 Rev. 3
Components
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Description Quantity/Size Part Number
Mouse B Lymphocyte Activation Antibody Cocktail; PE-Cy™7 CD25, PE CD69, and APC CD19 100 Tests (1 ea) 51-9003392
Mouse B Lymphocyte Activation Isotype Control; PE-Cy™7, PE, and APC 100 Tests (1 ea) 51-9003393
558063 Rev. 3
Citations & References
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Development References (19)

  1. Bendelac A, Matzinger P, Seder RA, Paul WE, Schwartz RH. Activation events during thymic selection. J Exp Med. 1992; 175(3):731-742. (Biology). View Reference
  2. Brandle D, Muller S, Muller C, Hengartner H, Pircher H. Regulation of RAG-1 and CD69 expression in the thymus during positive and negative selection. Eur J Immunol. 1994; 24(1):145-151. (Biology). View Reference
  3. 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. (Biology). View Reference
  4. 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
  5. 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
  6. Godfrey DI, Zlotnik A. Control points in early T-cell development. Immunol Today. 1993; 14(11):547-553. (Biology). View Reference
  7. Krop I, Shaffer AL, Fearon DT, Schlissel MS. The signaling activity of murine CD19 is regulated during cell development. J Immunol. 1996; 157(1):48-56. (Biology). View Reference
  8. Krop I, de Fougerolles AR, Hardy RR, Allison M, Schlissel MS, Fearon DT. Self-renewal of B-1 lymphocytes is dependent on CD19. Eur J Immunol. 1996; 26(1):238-242. (Biology). View Reference
  9. 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. (Biology). View Reference
  10. 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. (Biology). View Reference
  11. Marzio R, Jirillo E, Ransijn A, Mauel J, Corradin SB. Expression and function of the early activation antigen CD69 in murine macrophages. J Leukoc Biol. 1997; 62(3):349-355. (Biology). View Reference
  12. Nishimura T, Kitamura H, Iwakabe K, et al. The interface between innate and acquired immunity: glycolipid antigen presentation by CD1d-expressing dendritic cells to NKT cells induces the differentiation of antigen-specific cytotoxic T lymphocytes. Int Immunol. 2000; 12(7):987-994. (Biology). View Reference
  13. 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
  14. 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
  15. 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
  16. 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
  17. Yokoyama WM, Koning F, Kehn PJ, et al. Characterization of a cell surface-expressed disulfide-linked dimer involved in murine T cell activation. J Immunol. 1988; 141(2):369-376. (Biology). View Reference
  18. Yokoyama WM, Maxfield SR, Shevach EM. Very early (VEA) and very late (VLA) activation antigens have distinct functions in T lymphocyte activation. Immunol Rev. 1989; 109:153-176. (Biology). View Reference
  19. Ziegler SF, Ramsdell F, Alderson MR. The activation antigen CD69. Stem Cells. 1994; 12(5):456-465. (Biology). View Reference
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558063 Rev. 3

Please refer to Support Documents for Quality Certificates

Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described

Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims.  Comparisons are not made against non-BD technologies, unless otherwise noted.

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