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anti-Myeloperoxidase-C2 (FITC) and anti-Lactoferrin (PE) antibody pair

ABIN1741602 产品详细信息, 供应商: Log in to see
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适用

宿主
小鼠
克隆类型 (克隆位点)
单克隆 ()
标记
FITC,PE
应用范围
Flow Cytometry (FACS)
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原理 This product is optimised for use with FIX&PERM®.
品牌 FIX&PERM®
克隆位点 8E6-4C5
亚型 IgG1
特异性 Antibody MPO-C2 (clone 8E6) reacts with human myeloperoxidase (MPO) expressed by normal and malignant myelomonocytic cells. The LF mAb (clone 4C5) recognizes lactoferrin stored within secondary granules of postmitotic granulocyte-commited cells. In this COMBI-IC Reagent antibody 8E6 is conjugated to FITC, antibody 4C5 is conjugated to Phycoeythrin (PE).The sensitivity of MPO-C2/LF mAb is determined by staining well-defined blood samples from representative donors with serial-fold mAb dilutions 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 dilution expected to represent the least amount of mAb needed to detect an identical percentage of cells). In practice, 50 µL of leukocytes containing 10^7cells/mL are stained with 20 µL 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.
产品特性 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.
纯化方法 Purified by Affinity Chromatography
组件 COMBI IC Reagent: Mouse anti Myeloperoxidase-C2 (FITC) and Mouse anti Lactoferrin (PE)
Sub Type Cocktail
分子量 14 kDa
应用备注 Permeabilization and Staining Procedure - In combination with our Permeabilization Kit FIX&PERM- (ABIN1741575) intracellular MPO-C2 and LF 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 MPO-C2/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
说明

Myeloperoxidase (MPO) is a glycoprotein present in the azurophil (primary) granules of myeloid cells, which appears in the myeloblast stage of myeloid cell differentiation. MPO is he most common functional protein of myeloid cells and is involved in the inflammatory response. It helps to kill microbes by breaking down peroxide in the presence of halide ions, contributing to the bactericidal function of granulocytes. The primary translation product of MPO undergoes glycosylation with production of the 89 kDa heme-free apopro-MPO form followed by incorporation of heme and conversion into the enzymatically active pro-MPO form. Subsequently, pro-MPO becomes targeted to azurophil granules where final processing occurs to produce mature dimeric MPO consisting of the 59-64 kDa MPO α-chain and the 14 kDa MPO β-chain. 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 MPO and LF allows the distinction between mature and immature myelomonocytic cells. The MPO-C2/LF COMBI-IC reagent permits the identification and enumeration of immature and more mature myelomonocytic cell populations in normal and malignant human blood and bone marrow 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.

实验流程

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

限制 仅限研究用
缓冲液 PBS pH 7.2, 1 % BSA, 0.05 % sodium azide
储存液 Sodium azide
注意事项 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.
注意事项 Do not freeze and protect from prolonged exposure to light.
储存条件 4 °C
储存方法 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.
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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).

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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).

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Murao, Stevens, Ito, Huberman: "Myeloperoxidase: a myeloid cell nuclear antigen with DNA-binding properties." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 85, Issue 4, pp. 1232-6, 1988 (PubMed).

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