Description

Description: Flow cytometric analyses with monoclonal antibodies were so far mainly restricted to cell surface molecules. Intracellular structures such as cytoplasmic or nuclear enzymes, oncoproteins, cytokines, immunoglobulins etc. were largely excluded from such studies. Also excluded from flow cytometric studies were cytoplasmic localizations of well-established membrane molecules like CD3 and CD22, which, in their cytoplasmic form, are the most reliable lineage markers in undifferentiated leukemia. With the FIX&PERM® Kit flow cytometric analysis of intracellular antigens has become as easy as surface antigen studies. The only prerequisite is the availability of suitable antibody conjugates. Most of the available monoclonal antibody conjugates can be used with the FIX&PERM® Kit, some determinants are sensitive, however, to the fixation step involved. This and the optimal fixation time have to be tested for each reagent. 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

Application Notes

Biological fluids (blood, bone marrow, and others) must be collected under sterile conditions. Anticoagulation with EDTA or heparin is recommended. The samples should be stored at room temperature until used. For optimal results, samples should be processed and analyzed within 24 hours. Samples with high numbers of non-viable cells might cause false results, such cases require determination of cell viability with e.g. propidium iodide. All biological samples have to be handled with caution. Always consider them as potentially infective. Use appropriate precautions such as gloves, lab-coat, etc. Fixation, permeabilization and staining procedure: • 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 appropriate Nordic-MUbio 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. Comments: Special cases (diluted bone marrow samples, other samples containing low soluble protein) might benefit from replenishment with plasma components before the FIX&PERM® treatment in order to create a milieu, which more closely resembles the stuation in anti-coagulated blood. For that pupose addition of IgG preparations (e.g. Beriglobulin P, ZLB Behring, final concentration 10mg/ml) and human serum albumin (e.g. human albumin "Behring" 20% - infusion solution, final concentration 40mg/ml) is recommended.

Storage Note

FIX&PERM® Cell Fixation and Permeabilization Kit reagents should be stored and used at room temperature. Do not freeze. 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. If reagents are stored under any conditions other than those specified, the conditions must be verified by the user. Do not use reagents if a precipitate should form or discoloration occurs. If unexpected results are obtained which cannot be attributed to differences in laboratory procedures, please contact us.