Description

Description: The epitope recognized by antibody RFB4 is a 135 kDa type I integral membrane protein expressed by human B-cells. Precursor B-cells are surface-CD22 negative, but cytoplasmic CD22 positive. Mature B-lymphocytes express CD22 also on their surface. The RFB4 antibody permits the identification and enumeration of human B-cells using flow cytomet Results must be interpreted 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.

Additional Text

Gene Name

CD22

Gene ID

933

Uniprot ID

P20273

Purification

Purified

Antibody Clonality

Monoclonal

Application Notes

Staining Procedure for Surface CD22: 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 conjugate - 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 - 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 µl antibody conjugate 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 - 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 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 CD22: Permeabilization and Staining Procedure - In combination with our Permeabilization Kit FIX&PERM® (Cat. No. GAS-002) intracellular CD22 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 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 CD22 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

Storage Note

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.