Mouse anti CD3

Mouse anti CD3

• Background: UCHT1 is directed against Human CD3 – the multichain complex associated with the T-cell receptor. Precursor T-cells are surface CD3 negative but positive for cytoplasmic CD3. All mature T-cells are both cytoplasmic and surface CD3 positive. The UCHT1 antibody permits the identification and enumeration of normal and leukemic 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.
• CAS Number: 9007-83-4
• UniProt: P07766
• Host: Mouse
• Species Reactivity: Human
• Isotype: IgG1
• Clone: UCHT1
• Type: Primary Antibodies
• Applications: Flow Cytometry, IF
• Purification Method: Purified by Chromatography
• Assay Principle: Staining Procedure for Surface CD3: Direct Immunofluorescence (Staining Procedure) Nordic-MUbio fluorochrome labeled antibodies are designed for use with either whole blood or isolated mononuclear cell (MNC) preparations Proposed staining procedure for whole blood in short: - For each sample add 50 µL of EDTA anti-coagulated blood to a 3-5 mL tube - Add 20 µL of the appropriate Nordic-MUbio monoclonal antibody conjµgate - Incubate the tube for 15 minutes at 4°C or at room temperature in the dark - Add 100 µL Nordic-MUbio-LYSE (Cat.No. GAS-003) to each tube and incubate for 10 minutes at room temperature - Add 3-4 mL of destilled water and vortex, incubate for 5-10 minutes at room temperature - Centrifµge tube for 5 minutes at 300 g - Aspirate supernatant and resuspend pellet in 0.3 mL of sheath fluid - Analyze immediately or store samples at 2-8° C in the dark and analyze within 24 hours For “No-Wash” protocol please refer to www.nordicmubio.com Proposed staining procedure for MNC in short: - Carefully add 20 µL antibody conjµgate and 50-100 µL MNC to the bottom of a tube - Vortex at low speed for 1-2 seconds - Incubate for 15-30 minutes at 2-8°C or at room temperature - Centrifµge tubes for 5 minutes at 300 g - Remove supernatant, resuspend cells in 2-5 mL of phosphate buffered saline (PBS) and centrifµge cells again for 5 minutes at 300 g - Remove supernatant and resuspend cells in sheath fluid for immediate analysis or resuspend cells in 0.5 mL 1 % formaldehyde and store them at 2-8°C in the dark. - Analyze fixed cells within 24 hours Indirect Immunofluorescence (Staining Procedure) - Mix 20 µL Nordic-MUbio purified antibody with 50 µL whole blood or MNC suspension - Incubate for 15 minutes at 2-8°C - Wash cells with phosphate buffered saline (PBS) - Add to cell pellet 20 µL of affinity purified, fluorochrome labeled F(ab’)2 anti mouse Ig antibodies - Incubate for 15 minutes at 2-8°C - Wash cells with phosphate buffered saline (PBS) or proceed as described for direct staining Staining Procedure for Cytoplasmatic CD3: Permeabilization and Staining Procedure - In combination with our Permeabilization Kit FIX,PERM® (Cat. No. GAS-002) intracellular 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 5mL tube - Add 100 µL of Reagent A (Fixation Medium, stored and used at room temperature) - Incubate for 15 minutes at room temperature - Add 5mL phosphate buffered saline and centrifµge 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 CD3 monoclonal antibody conjµgate - 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.
• Form: Purified by Chromatography, Storage buffer: PBS pH 7,2, 1% BSA, 0,05% NaN3
• Precautions: 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.
• References & Citations: 1. Paietta, E. (2003) Best Pract Res Clin Haematol 16, 671-83. _x000D_ 2. Braylan, R. C., Orfao, A., Borowitz, M. J. , Davis, B. H. (2001) Cytometry 46, 23-7. _x000D_ 3. Lanza, F., Latorraca, A., Moretti, S., Castagnari, B., Ferrari, L. , Castoldi, G. (1997) Cytometry 30, 134-44. _x000D_ 4. Groeneveld, K., te Marvelde, J. G., van den Beemd, M. W., Hooijkaas, H. , van Dongen, J. J. (1996) Leukemia 10, 1383-9. _x000D_ 5. Catovsky, D., Matutes, E., Buccheri, V., Shetty, V., Hanslip, J., Yoshida, N. , Morilla, R. (1991) Ann Hematol 62, 16-21. _x000D_ 6. Janossy, G., Coustan-Smith, E. , Campana, D. (1989) Leukemia 3, 170-81. _x000D_ 7. Clevers, H., Alarcon, B., Wileman, T. , Terhorst, C. (1988) Annu Rev Immunol 6, 629-62. _x000D_ 8. Wering, E. R. , Terhorst, C. (1988) Blood 71, 603-12. _x000D_ 9. Rani, S., De Oliveira, M. S. , Catovsky, D. (1988) Hematol Pathol 2, 73-8. _x000D_ 10. van der Schoot, C. E., von dem Borne, A. E. , Tetteroo, P. A. (1987) Acta Haematol 78 Suppl 1, 32-40. _x000D_ 11. van Dongen, J. J., Krissansen, G. W., Wolvers-Tettero, I. L., Comans-Bitter, W. M., Adriaansen, H. J., Hooijkaas, H., van Campana, D., Thompson, J. S., Amlot, P., Brown, S. , Janossy, G. (1987) J Immunol 138, 648-55. _x000D_ 12. Burns, G. F., Boyd, A. W. , Beverley, P. C. (1982) J Immunol 129, 1451-7 Beverley, P. C., Linch, D. , Callard, R. E. (1981) Haematol Blood Transfus 26, 309-13.
• Storage Conditions: 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. If a slight precipitation occurs upon storage, this should be removed by centrifugation; It will not affect the performance or the concentration of the product 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.