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Mouse T Lymphocyte Activation Antibody Cocktail, with Isotype Control PE-Cy™7 CD25, PE CD69, and APC CD3e

Mouse T Lymphocyte Activation Antibody Cocktail, with Isotype Control PE-Cy™7 CD25, PE CD69, and APC CD3e

(RUO)
Mouse T Lymphocyte Activation Antibody Cocktail, with Isotype Control  PE-Cy™7 CD25, PE CD69, and APC CD3e

Identification of activated T lymphocytes using Mouse T Lymphocyte Activation Antibody Cocktail, with Isotype Control. BALB/c splenocytes were activated in culture for 48 hours on plate-bound Purified Hamster Anti-Mouse CD3e (Clone 500A2; Cat. No. 553238) and scatter plots were used to select either activated lymphoblasts (Left and Middle Panels) or resting (unactivated) lymphocytes (Right Panels) for data analysis.  The activated splenocytes were stained with either Mouse T Lymphocyte Activation Isotype Control (Left Panels) or Mouse T Lymphocyte Activation Antibody Cocktail (Middle Panels). Resting splenocytes were stained with Mouse T Lymphocyte Activation Antibody Cocktail (Right Panels) or Mouse T Lymphocyte Activation Isotype Control (not shown). The two-color contour plots display the CD3e+ activated lymphoblasts which have upregulated CD25 (Middle Top Panel) and CD69 (Middle Bottom Panel) antigen expression. Flow cytometric analysis was performed on a BD FACSCalibur™ flow cytometry system.

Identification of activated T lymphocytes using Mouse T Lymphocyte Activation Antibody Cocktail, with Isotype Control. BALB/c splenocytes were activated in culture for 48 hours on plate-bound Purified Hamster Anti-Mouse CD3e (Clone 500A2; Cat. No. 553238) and scatter plots were used to select either activated lymphoblasts (Left and Middle Panels) or resting (unactivated) lymphocytes (Right Panels) for data analysis.  The activated splenocytes were stained with either Mouse T Lymphocyte Activation Isotype Control (Left Panels) or Mouse T Lymphocyte Activation Antibody Cocktail (Middle Panels). Resting splenocytes were stained with Mouse T Lymphocyte Activation Antibody Cocktail (Right Panels) or Mouse T Lymphocyte Activation Isotype Control (not shown). The two-color contour plots display the CD3e+ activated lymphoblasts which have upregulated CD25 (Middle Top Panel) and CD69 (Middle Bottom Panel) antigen expression. Flow cytometric analysis 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_396932


Description

The Mouse T Lymphocyte Activation Antibody Cocktail is a three-color reagent designed to identify activated T lymphocytes by direct immunofluorescent staining with flow cytometric analysis. This cocktail consists of the following antibody mixture:  PE-Cy7 rat anti-mouse CD25 (clone PC61), PE hamster anti-mouse CD69 (clone H1.2F3), and APC hamster anti-mouse CD3e (clone 145-2C11). 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 expressed also on thymocytes that are undergoing positive selection. The 145-2C11 antibody reacts with the 25-kDa echain of the T-cell receptor-associated CD3 complex, which is expressed on thymocytes, mature T lymphocytes, and NK-T cells of all mouse strains tested. The three antibodies have been titrated and pre-diluted, mixed together, and formulated for optimal staining performance. The Mouse T Lymphocyte Activation Isotype Control contains equivalent concentrations of fluorochrome- and isotype-matched negative-control immunoglobulins.

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 T lymphocytes. Additional fluorochrome-labeled reagents may be combined with the Mouse T Lymphocyte Activation Antibody Cocktail, and the Mouse T Lymphocyte Activation Isotype Control, to further characterize activated T-cell subpopulations.

The Mouse T Lymphocyte Activation Isotype Control contains equivalent concentrations of fluorochrome- and isotype-matched negative-control immunoglobulin consisting of the following: PE-Cy™7 rat IgG1, λ (clone A110-1), PE Armenian hamster IgG1, λ (clone G235-2356) and APC Armenian hamster IgG1, κ (clone A19-3).

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 R-PE under optimum conditions, and unconjugated antibody and free PE were removed. The antibody was conjugated with PE-Cy7 under optimum conditions, and unconjugated antibody and free PE-Cy7 were removed. The antibody was conjugated to APC under optimum conditions, and unconjugated antibody and free APC were removed.

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. An isotype control should be used at the same concentration as the antibody of interest.
  3. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  4. 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.
  5. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  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. 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.
  8. This APC-conjugated reagent can be used in any flow cytometer equipped with a dye, HeNe, or red diode laser.
  9. 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).
  10. Cy is a trademark of GE Healthcare.
  11. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
557908 Rev. 4
Components
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Description Quantity/Size Part Number
Mouse T Lymphocyte Activation Antibody Cocktail PE-Cy™7 CD25, PE CD69, and APC CD3e 100 Tests (1 ea) 51-9002235
Mouse T Lymphocyte Subset Isotype Control PE-Cy™7 Rat IgG1, λ, PE Hamster IgG1, λ and APC Hamster IgG1, κ 100 Tests (1 ea) 51-9002237
557908 Rev. 4
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. Leo O, Foo M, Sachs DH, Samelson LE, Bluestone JA. Identification of a monoclonal antibody specific for a murine T3 polypeptide. Proc Natl Acad Sci U S A. 1987; 84(5):1374-1378. (Biology). View Reference
  8. 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
  9. 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
  10. Maruoka H, Ikarashi Y, Shinohara K, et al. A novel monoclonal antibody permitting recognition of NKT cells in various mouse strains. Biochem Biophys Res Commun. 1998; 242(2):413-418. (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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557908 Rev. 4

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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.