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 aellet 100 µl Reagent B (Permeabilization Medium) and 20 µl of the appropriate 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 2-8C 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 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)
Flow cytrometric analysis of normal bone marrow (BM) after fixation and pemeabilization using orb1972402, followed by immunostaining for lactoferrin and MPO-C2.
Flow cytrometric analysis of bone marrow (BM) from a leukemia patient after fixation and pemeabilization using orb1972402, followed by immunostaining for lactoferrin and MPO-C2.
Immunostaining with CD22-PE conjugate of undifferentiated leukemia cells of B-ALL type.
Immunostaining with CD3-PE conjugate of surface CD3-negative undifferentiated leukemia cells of T-ALL type.
Immunostaining with anti MPO-FITC conjugate of undifferentiated leukemia cells of AML type.
Forward and side scatter pattern of a normal blood sample treated with orb1972402.
Double labeling of a normal blood sample treated with orb1972402, and immunostained for Lactoferrin (PE) and MPO-C2 (FITC).
Double labeling of a normal blood sample treated with orb1972402, and immunostained for CD3 (PE) and MPO-C2 (FITC).
Double labeling of a normal blood sample treated with orb1972402, and immunostained for Lactoferrin (PE) and Lysozyme (FITC).
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