anti-Lysozyme (FITC) and anti-Lactoferrin (PE) antibody pair

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Antigen
Reactivity
Human
Host
Mouse
Clonality (Clone)
Monoclonal ()
Conjugate
FITC,PE
Application
Flow Cytometry (FACS)
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Purpose This product is optimised for use with FIX&PERM®.
Brand FIX&PERM®
Clone LZ-2-4C5
Isotype IgG1
Specificity The anti-Lysozyme Antibody (clone LZ-2) reacts with intracellular human lysozyme/muramidase expressed by virtually all myelomonocytic cells, macrophages and their precursors. The LF mAb (clone 4C5) recognizes lactoferrin stored within secondary granules of postmitotic granulocyte-commited cells.In this COMBI-IC Reagent antibody LZ-2 is conjugated to FITC, antibody 4C5 is conjugated to Phycoeythrin (PE).The sensitivity y of LZ/LF mAb is determined by staining well-defined blood samples from representative donors with serial-fold mAbdilutions to obtain a titration curve that allows relating the mAb concentration to the percentage of stained cells and geometric MFI (mean fluorescence intensity). For this purpose, a mAb-concentration range is selected to include both the saturation point (i.e. the mAb dilution expected to bind all epitopes on the target cell) and the detection threshold (i.e. the mAb dilutionexpected to represent the least amount of mAb needed to detect an identical percentage of cells). In practice, 50 µL of leukocytes containing 10^7 cells/mL are stained with 20µL mAb of various dilutions to obtain a titration curve and to identify the saturation point and detection threshold. The final concentration of the product is then adjusted to be at least 3-fold above the detection threshold. In addition and to control lot-to-lot variation, the given lot is compared and adjusted to fluorescence standards with defined intensity.
Characteristics 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.
Purification Purified by Affinity Chromatography
Components COMBI IC Reagent: Mouse anti Lysozyme (FITC) and Mouse anti Lactoferrin (PE)
Sub Type Cocktail
Molecular Weight 14 kDa
Application Notes Permeabilization and Staining Procedure- In combination with our Permeabilization Kit FIX&PERM- (ABIN1741575) intracellular Lysozyme and Lactoferrin 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 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 LZ/LF COMBI-IC 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.
Comment

Lysozyme (LZ) is a cationic antimicrobial peptide of 14 kDa. Lysozyme is stored in primary but predominantly in specific (secondary) granules of neutrophils. It cleaves peptidoglycan constituents of the bacterial cell wall and can bind LPS. The epitope recognized by antibody LZ-2 is expressed by virtually all myeloid cells including normal and malignant granulocytes and monocytes. In normal myelopoiesis LZ can first be detected at the myeloblast stage where it appears somewhat later than MPO expression. Lactoferrin (LF) is an iron-binding protein with bactericidal and bacteriostatic activity, which is stored within the secondary granules of granulocytes. LF expression is restricted to the post-mitotic maturation compartment of the granulocytic lineage, starting from the myelocyte stage. Normal and malignant myeloblasts are LF negative. The combined staining for Lysozyme and Lactoferrin allows the distinction between mature and immature myelomonocytic cells. The LZ/LF COMBI-IC reagent permits the identification and enumeration of myeloid cells in normal and malignant human blood and bone marrow samples 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. 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

Assay Procedure

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

Restrictions For Research Use only
Buffer PBS pH 7.2, 1 % BSA, 0.05 % sodium azide
Preservative Sodium azide
Precaution of Use This reagent contains sodium azide. To avoid the development of hazardous conditions, reagents containing azide should be diluted in running water prior to be discarded. Similar to the work with other biological products, proper handling procedures are recommended.
Handling Advice Do not freeze and protect from prolonged exposure to light.
Storage 4 °C
Storage Comment These reagents should be stored at 2-8°C 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.
Product cited in: Strobl, Knapp: "Myeloid cell-associated lysosomal proteins as flow cytometry markers for leukocyte lineage classification." in: Journal of biological regulators and homeostatic agents, Vol. 18, Issue 3-4, pp. 335-9, 2005 (PubMed).

Teng, Gladwell, Beard, Walmer, Teng, Brenner: "Lactoferrin gene expression is estrogen responsive in human and rhesus monkey endometrium." in: Molecular human reproduction, Vol. 8, Issue 1, pp. 58-67, 2001 (PubMed).

Braylan, Orfao, Borowitz, Davis: "Optimal number of reagents required to evaluate hematolymphoid neoplasias: results of an international consensus meeting." in: Cytometry, Vol. 46, Issue 1, pp. 23-7, 2001 (PubMed).

Koníková, Glasová, Kusenda, Babusíková: "Intracellular markers in acute myeloid leukemia diagnosis." in: Neoplasma, Vol. 45, Issue 5, pp. 282-91, 1999 (PubMed).

Oehler, Majdic, Pickl, Stöckl, Riedl, Drach, Rappersberger, Geissler, Knapp: "Neutrophil granulocyte-committed cells can be driven to acquire dendritic cell characteristics." in: The Journal of experimental medicine, Vol. 187, Issue 7, pp. 1019-28, 1998 (PubMed).

Lanza, Latorraca, Moretti, Castagnari, Ferrari, Castoldi: "Comparative analysis of different permeabilization methods for the flow cytometry measurement of cytoplasmic myeloperoxidase and lysozyme in normal and leukemic cells." in: Cytometry, Vol. 30, Issue 3, pp. 134-44, 1997 (PubMed).

Madrigal, Belich, Benjamin, Little, Hildebrand, Mann, Parham: "Molecular definition of a polymorphic antigen (LA45) of free HLA-A and -B heavy chains found on the surfaces of activated B and T cells." in: The Journal of experimental medicine, Vol. 174, Issue 5, pp. 1085-95, 1991 (PubMed).

Srivastava, Rado, Bauerle, Broxmeyer: "Regulation of human bone marrow lactoferrin and myeloperoxidase gene expression by tumor necrosis factor-alpha." in: Journal of immunology (Baltimore, Md. : 1950), Vol. 146, Issue 3, pp. 1014-9, 1991 (PubMed).

Catovsky, Matutes, Buccheri, Shetty, Hanslip, Yoshida, Morilla: "A classification of acute leukaemia for the 1990s." in: Annals of hematology, Vol. 62, Issue 1, pp. 16-21, 1991 (PubMed).

Rado, Bollekens, St Laurent, Parker, Benz: "Lactoferrin biosynthesis during granulocytopoiesis." in: Blood, Vol. 64, Issue 5, pp. 1103-9, 1984 (PubMed).