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ApoFlowEx® FITC Kit

ABIN3072911 产品详细信息, 供应商: Log in to see
应用范围
Flow Cytometry (FACS)
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原理 The ApoFlowEx® FITC Kit is intended for differentiation of early apoptotic, necrotic and viable cells.
品牌 ApoFlowEx®
样品类型 Cell Samples
组件
  • Annexin V-FITC
    1 vial. 0.5 ml of Ready-toUse solution. No reconstitution is necessary. Reagent is provided in stabilizing phosphate buffered saline (PBS) solution containing 15mM sodium azide. Sufficient for 100 tests.
  • Propidium Iodide
    1 vial. 0.5 ml of Readyto-Use solution in deionized water (0.1 mg/ml). Sufficient for 100 tests.
  • Annexin V Binding Buffer (10x)
    1 vial. 5 ml of 10x concentrated filter-sterilized solution (0.1M HEPES/NaOH, pH 7.4, 1.4M NaCl, 25mM CaCl2). Dilute 10x in deionized water prior to use.
试剂未包括
  • Suitable 5 mL test tubes for cell staining (e.g. 12 x 75 mm)
  • Ultrapure demineralized water
  • PBS (Phosphate Buffered Saline)
  • Automatic pipettes with disposable tips
  • Vortex mixer
  • Centrifuge with rotor suitable for test tubes
  • Flow cytometer - blue laser excitation at 488 nm, light emission at 525 nm (FITC) and 617 nm (Propidium iodide)
背景 Apoptosis is a regulated cell death process characterized by morphological and biochemical features occurring at different stages. The translocation of phosphatidylserine (PS) from the inner side of the plasma membrane to the outer layer is one of many plasma membrane alterations during apoptosis. By this process PS becomes exposed on the external surface of the cell. Detection of such membrane changes by Annexin V binding to PS has been suggested as a suitable assay for early apoptotic cells.

Annexin V is a 35 kDa intracellular protein. It belongs to the family of proteins that bind to phosphatidylserine (PS) under a Ca2+-dependent conditions. Fluorescently labeled Annexin V can be applied, with appropriate buffer (Annexin V Binding Buffer), for direct quantification of apoptotic cells using suitable protocols. Since PS membrane translocation also occurs during necrosis, Annexin V is not an absolute marker of apoptosis. It is often used simultaneously with appropriate viability evaluation dyes such as propidium iodide (PI) to counterstain dead cells. PI is an intercalating agent and a fluorescent molecule. When PI binds to nucleic acids, the fluorescence excitation maximum is 535 nm and the emission maximum is 617 nm. Propidium Iodide passes through the plasmatic membrane of late apoptotic cells and necrotic cells and bind to chromosomal DNA. Plasmatic membrane of viable and early apoptotic cells is impermeable for PI.
应用备注 Suggested Cell Number: 2 - 5 x105 cells/ ml
  • The flow cytometer should be calibrated on a routine basis using fluorescent microbeads to ensure stable sensitivity of detectors.
  • Do not use reagents after expiration date stated on vial labels.
  • Avoid contamination of the reagent.
  • Avoid Annexin V - FITC prolonged exposure to light.
  • Avoid Propidium Iodide prolonged exposure to light.
  • Any non-performance of assay procedure may produce false results.
样本量 100 μL
试剂准备

Preparation of reagents before measurement

  • The Annexin V Binding Buffer (10x)
    The Annexin V Binding Buffer (10x) is a 10x concentrated and must be diluted with deionized water prior to use to prepare 1 x Annexin V Binding Buffer. Use fresh solution for each experiment.

实验流程
  1. Harvest cells intended for analysis by centrifugation (different cells may need different centrifugation conditions), discard supernatant. Resuspend cell pellet in cold PBS and wash cells by gentle shaking or by up-anddown mixing in a pipette tip. Re-centrifuge washed cells again and discard supernatant.
  2. Resuspend cell pellet in 1 x Annexin V Binding Buffer and adjust cell density to 2 - 5 x 105 cells/ ml, preparing a sufficient volume of cell suspension (100 μL per assay).
  3. Add 5 μL of Annexin V - FITC and 5 μL of Propidium Iodide to each 100 μL of cell suspension and mix gently.
  4. Incubate stained cells for 15 minutes in dark at room temperature.
  5. After the incubation period, centrifuge cells and resuspend pellet in 100 μL of 1 x Annexin V Binding Buffer (or in an appropriate volume according to a method of sample acquisition)
  6. Analyze the stained cells by flow cytometry as soon as possible.

Analyze stained samples using flow cytometer. Acquire at least 5.000 - 10.000 cells per sample. Make compensation of fluorescent signals prior or after data acquisition if needed. Visualize measured data from cells in the side-scatter (SSC) versus forward-scatter (FSC) dot-plot. Set the gate around cells. Then bring the gated cells to dot-plot diagram where the X-axis represents fluorescence intensity in FITC channel (FL1) and Y-axis represents PI channel (FL4). Set appropriate gates to calculate the percentage of viable, apoptotic and necrotic cells. Apoptotic cells exhibit high intensity of fluorescence in FITC channel.
限制 仅限研究用
储存条件 4 °C