- Flow Cytometry (FACS)
- NM-LYSE Reagent is designed for use with all commercially available flow cytometers. Alignment and compensation should be performed according to manufacturer´s instructions The quality of each NM-LYSE Lot is determined by lysing red blood cells of well defined blood samples from representative donors and subsequent comparison of forward and side scatter characteristics of obtained leukocytes.
Mildly fixes cells, preserving their flow cytometric scatter characteristics
Allows simultaneous characterisation of both intracellular and cell surface markers
Rapid technique - whole procedure can be carried out in less than one hour, ready
for immediate analysis or storage for 24 hours
Stringent QC procedures - the quality of each lot is determined using well-defined
blood samples and subsequent comparison of scatter characteristics of obtained
leukocyte populations, ensuring consistent and reliable results lot after lot
A range of intracellular antibodies with optimised protocols for use with FIX&PERM®
FIX&PERM® is a simple procedure making use of two reagents. Reagent A gently fixes cells, while Reagent B permeabilizes them. The specific formulations reduce background and allow simultaneous addition of permeabilization medium and fluorochrome labelled antibodies, allowing staining of intracellular structures such as cytoplasmic or nuclear enzymes, oncoproteins, cytokines, immunoglobulins, etc.
- No-Wash Staining and Lysing Procedure - For each sample add 50 µL of EDTA anti-coagulated blood to a 3-5 mL tube - Add 20 µL of the appropriate monoclonal antibody conjugate - Incubate the tube for 15 minutes at 4°C or at room temperature in the dark - Add 100 µL NM-LYSE to each tube and incubate for 10 minutes at room temperature - Add 1 mL of destilled water and vortex, incubate for 5-10 minutes at room temperature - Analyze immediately or store samples at 2-8° C in the dark and analyze within 24 hours Wash Staining and Lysing Procedure - For each sample add 50 µL of EDTA anti-coagulated blood to a 3-5 mL tube - Add 20 µL of the appropriate monoclonal antibody conjugate - Incubate the tube for 15 minutes at 4°C or at room temperature in the dark - Add 100 µL NM-LYSE 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 Wash- or No-Wash Lysing Procedures with Whole Blood or Marrow Samples NM-LYSE is a premixed, ready to use lysing solution fomulated for lysing erythrocytes following monoclonal antibody staining of whole blood. Treatment with this reagent simultaneously leads to lysis of red blood cells and fixation of white cells. Morphological scatter characteristics of leukocytes remain intact. NM-LYSE can be used with or without sample washing. NM-LYSE is suitable for the analysis of normal and malignant leukocyte populations derived from various human biological samples (blood, bone marrow and others) 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. Flow cytometric analyses with monoclonal antibodies were so far restricted to leukocyte populations, which had been separated from erythrocytes before staining and/or analysis. Instead, whole blood staining methods allow for a rapid and accurate determination of cellular subpopulations in non-separated biological samples. This is not only time saving but reduces also the probability of an unintended loss of distinct cellular populations due to e.g. commonly used differential centrifugation procedures. With the NM-LYSE reagent flow cytometric analysis of whole blood has become as easy and accurate as the analysis of separated cell populations.
Procedure: For each sample to be analysed use 50 µL of whole blood, bone marrow or mononuclear cell suspension in a 5 mL tube
Add 100 µL of Reagent A (Fixation Medium, stored and used at room temperature)
Incubate for 15 minutes at room temperature
Add 5 mL 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 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 re-suspend cells in sheath fluid for immediate analysis or re-suspend cells in 0.5 mL 1.0 % formaldehyde and store at 2-8°C in the dark
Analyse fixed cells within 24 hours
- Nur für Forschungszwecke einsetzbar
- NM-LYSE contains fomaldehyde. Formaldehyde is toxic, allergenic and a suspected carcinogen. Avoid contact with eyes, skin and clothing. Proper handling procedures are recommended.
- 4 °C
- Informationen zur Lagerung
- NM-LYSE reagent should be stored and used at room temperature. 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. Do not use reagent 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
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: "Comparison between a lyse-and-then-wash method and a lyse-non-wash technique for the enumeration of CD34+ hematopoietic progenitor cells." in: Cytometry, Vol. 34, Issue 6, pp. 264-71, (1999) (PubMed).
: "Comparative analysis of whole blood lysis methods for flow cytometry." in: Cytometry, Vol. 30, Issue 3, pp. 124-33, (1997) (PubMed).
: "Quantification of CD34+ cells: comparison of methods." in: Transfusion, Vol. 37, Issue 8, pp. 775-84, (1997) (PubMed).
- Serum proteins modified by neutrophil-derived oxidants as mediators of neutrophil stimulation." in: Journal of immunology (Baltimore, Md. : 1950), Vol. 167, Issue 1, pp. 451-60, (2001) (PubMed).