Mouse anti CD3

Nordic-MUbio

Mouse anti CD3

7

The CD3 mAb (clone UCHT1) recognizes cytoplasmic CD3 epsilon in precursor T-cells and cytoplasmic and surface CD3 epsilon in mature T-lymphocytes. The sensitivity of UCHT1 mAb is determined by staining well-defined blood samples from representative donors

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 microl of EDTA anti-coagulated blood to a 3-5 ml tube - Add 20 microl of the appropriate Nordic-MUbio monoclonal antibody conjugate - Incubate the tube for 15 minutes at 4°C or at room temperature in the dark - Add 100 microl 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 - Centrifuge 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 microl antibody conjugate and 50-100 microl 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 - Centrifuge tubes for 5 minutes at 300 g - Remove supernatant, resuspend cells in 2-5 ml of phosphate buffered saline (PBS) and centrifuge 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 microl Nordic-MUbio purified antibody with 50 microl 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 microl 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 microl of whole blood, bone marrow or mononuclear cell suspension in a 5ml tube - Add 100 microl of Reagent A (Fixation Medium, stored and used at room temperature) - Incubate for 15 minutes at room temperature - Add 5ml phosphate buffered saline and centrifuge cells for 5 minutes at 300 g - Remove supernatant and add to cell pellet 100 microl Reagent B (Permeabilization Medium) and 20 microl of the CD3 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.

Flow Cytometry, ImmunoFluorescence

Flow Cytometry;Immunofluoresence;Immunofluoresence

Primary antibodies

Research Use Only

Monoclonal

UCHT1

CD3E

CD3e molecule

CD3e antigen, epsilon polypeptide (TiT3 complex); CD3e molecule, epsilon (CD3-TCR complex); CD3-epsilon; IMD18; T3E; T-cell antigen receptor complex, epsilon subunit of T3; T-cell surface antigen T3/Leu-4 epsilon chain; T-cell surface glycoprotein CD3 epsilon chain; TCRE

Mouse

IgG1

T-cell surface glycoprotein CD3 epsilon chain

Human

Human

2°C to 8°C

12352203