COMBI IC Reagent: Mouse anti Myeloperoxidase-C2 (FITC) and Mouse anti CD3 (PE)


Catalogue number: GIC-213

Clone8E6 and UCHT1
IsotypeIgG1
Product Type Primary Antibodies
Units1 ml
HostMouse
Species reactivity Human
Application Flow cytometry
Background
Myeloperoxidase (MPO) is a glycoprotein present in the azurophil (primary) granules of myeloid cells, which appears in the myeloblast stage of myeloid cell differentiation. MPO is he most common functional protein of myeloid cells and is involved in the inflammatory response. It helps to kill microbes by breaking down peroxide in the presence of halide ions, contributing to the bactericidal function of granulocytes. The primary translation product of MPO undergoes glycosylation with production of the 89 kDa heme-free apopro-MPO form followed by incorporation of heme and conversion into the enzymatically active pro-MPO form. Subsequently, pro-MPO becomes targeted to azurophil granules where final processing occurs to produce mature dimeric MPO consisting of the 59-64 kDa MPO a-chain and the 14 kDa MPO b-chain. Precursor T-cells are surface CD3 negative but positive for cytoplasmatic CD3. All mature T cells are both cytoplasmic and surface CD3 positive. The combined staining for MPO and CD3 allows the distinction of cells derived form the myeloid lineage that are generally MPO positive, from immature and mature T lymphocytes during normal and malignant hematopoiesis. The MPO-C2/CD3 COMBI-IC reagent permits the identification and enumeration of normal and malignant human blood and bone marrow cells using flow cytometry. Results must be put within the context of other diagnostic tests as well as the clinical history of the patient by a certified professional before final interpretation. Analyses performed with this antibody should be paralleled by positive and negative controls. If unexpected results are obtained which cannot be attributed to differences in laboratory procedures, please contact us.

Product
1ml of FITC-conjugated anti Myeloperoxidase-C2 (clone 8E6) and PE-conjugated anti CD3 (cloneUCHT1) in PBS pH 7.2, 1% BSA, and 0.05% NaN3, approximately 50 tests.

Applications
Permeabilization and Staining Procedure - In combination with our Permeabilization Kit FIX&PERM (Cat. No. GAS-002) intracellular MPO-C2 and CD3 can be easily stained in cell suspensions - For each sample to be analyzed add 50 µl of whole blood, bone marrow or mononuclear cell suspension in a 5 ml tube - Add 100 µl of Reagent A (Fixation Medium, stored and used at room temperature) - Incubate for 15 minutes at room temperature - Add 5 ml phosphate buffered saline and centrifuge cells for 5 minutes at 300 g - Remove supernatant and add to cell pellet 100 µl Reagent B (Permeabilization Medium) and 20 µl of the MPO-C2/CD3 COMBI-IC monoclonal antibody conjugate - Vortex at low speed for 1-2 seconds - Incubate for 15 minutes at room temperature - Wash cells with phosphate buffered saline as described above -Remove supernatant and resuspend cells in sheath fluid for immediate analysis or resuspend cells in 0.5 ml 1.0 % formaldehyde and store them at 2-8°C in the dark. Analyze fixed cells within 24 hours

Specificity
Antibody MPO-C2 (clone 8E2) reacts with human myeloperoxidase (MPO) expressed by normal and malignant myelomonocytic cells. The CD3 mAb (clone UCHT1) recognizes cytoplasmatic CD3 epsilon in precursor T-cells and cytoplasmic and surface CD3 epsilon in mature T-lymphocytes. In this COMBI-IC Reagent antibody 8E6 is conjugated to FITC, antibody UCHT1 is conjugated to Phycoeythrin (PE).

Storage
Nordic-MUbio monoclonal antibody reagents contain optimal concentrations of affinity-purified antibody. For stability reasons this monoclonal antibody solution contains sodium azide. These reagents should be stored at 2-8°C (DO NOT FREEZE!) and protected from prolonged exposure to light. Stability of the reagent: Please refer to the expiry date printed onto the vial. The use of the reagent after the expiration date is not recommended.

References
1. Andersson, E., Hellman, L., Gullberg, U. & Olsson, I. (1998) J Biol Chem 273, 4747-53.
2. Beverley, P. C., Linch, D. & Callard, R. E. (1981) Haematol Blood Transfus 26, 309-13
3. Braylan, R. C., Orfao, A., Borowitz, M. J. & Davis, B. H. (2001) Cytometry 46, 23-7.
4. Campana, D., Thompson, J. S., Amlot, P., Brown, S. & Janossy, G. (1987) J Immunol 138, 648-55.
5. Catovsky, D., Matutes, E., Buccheri, V., Shetty, V., Hanslip, J., Yoshida, N. & Morilla, R. (1991) Ann Hematol 62, 16-21.
6. Cowland, J. B. & Borregaard, N. (1999) J Leukoc Biol 66, 989-95.
7. Cramer, E., Pryzwansky, K. B., Villeval, J. L., Testa, U. & Breton-Gorius, J. (1985) Blood 65, 423-32
8. Groeneveld, K., te Marvelde, J. G., van den Beemd, M. W., Hooijkaas, H. & van Dongen, J. J. (1996) Leukemia 10, 1383-9.
9. Imamura, N. (1998) Am J Hematol 58, 241-3.
10. Janossy, G., Coustan-Smith, E. & Campana, D. (1989) Leukemia 3, 170-81.
11. Knapp, W., Majdic, O. & Strobl, H. (1993) Recent Results Cancer Res 131, 31-40.
12. Koeffler, H. P., Ranyard, J. & Pertcheck, M. (1985) Blood 65, 484-91.
13. Konikova, E., Glasova, M., Kusenda, J. & Babusikova, O. (1998) Neoplasma 45, 282-91.
14. Murao, S., Stevens, F. J., Ito, A. & Huberman, E. (1988) Proc Natl Acad Sci U S A 85, 1232-6.
15. Nakase, K., Sartor, M. & Bradstock (1998) Cytometry 34, 198-202.
16. Nauseef, W. M. (1990) Hematol Pathol 4, 165-78.
17. Nauseef, W. M., Olsson, I. & Arnljots, K. (1988) Eur J Haematol 40, 97-110.
18. Paietta, E. (2003) Best Pract Res Clin Haematol 16, 671-83.
19. Rani, S., De Oliveira, M. S. & Catovsky, D. (1988) Hematol Pathol 2, 73-8.
20. Srivastava, C. H., Rado, T. A., Bauerle, D. & Broxmeyer, H. E. (1991) J Immunol 146, 1014-9
21. Strobl, H. & Knapp, W. (2004) J Biol Regul Homeost Agents 18, 335-9.
22. Strobl, H., Takimoto, M., Majdic, O., Fritsch, G., Scheinecker, C., Hocker, P. & Knapp, W. (1993) Blood 82, 2069-78.
23. Tsuruta, T., Tani, K., Hoshika, A. & Asano, S. (1999) Leuk Lymphoma 32, 257-67.
24. Vainchenker, W., Villeval, J. L., Tabilio, A., Matamis, H., Karianakis, G., Guichard, J., Henri, A., Vernant, J. P., Rochant, H. & Breton-Gorius, J. (1988) Leukemia 2, 274-81.
25. van der Schoot, C. E., Daams, G. M., Pinkster, J., Vet, R. & von dem Borne, A. E. (1990) Br J Haematol 74, 173-8.
26. van der Schoot, C. E., von dem Borne, A. E. & Tetteroo, P. A. (1987) Acta Haematol 78 Suppl 1, 32-40.

Caution
For professional users only. This reagent contains sodium azide. To avoid the development of hazardous conditions, reagents containing azide should be diluted in running water prior to be discarded. Similar to the work with other biological products, proper handling procedures are recommended.