NKFlowEx® Kit

ABIN3072915 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 NKFlowEx® Kit is intended for the detection of CD69 activation marker of Natural killer (NK) cells after their activation with variety of stimuli in human heparinized whole blood using flow cytometry.
品牌 NKFlowEx®
样品类型 Blood Cells
组件
  • Staining Reagent
    vial containing 0.5 ml of premixed antibody cocktail: anti-CD45 (MEM-28), FITC labeled / anti-CD16 (3G8) + CD56 (LT56), PE labeled / anti-CD3 (UCHT-1), PE-Cy™5 labeled / anti-CD69 (FN50), PE-Cy™7 labeled.
  • Stimulation Control
    2 vials containing lyophilized PWM (Pokeweed mitogen), 1 vial is intended for stimulation of 13 positive controls.
  • 10x Lysing Solution
    10 ml.
试剂未包括
  • Suitable 5 mL test tubes for blood staining (e.g. 12 x 75 mm)
  • Culture medium suitable for cultivation of blood samples
  • 96 well tissue culture test plates
  • Automatic pipettes with disposable tips
  • Vortex mixer
  • Cell/tissue culture incubator (CO2, 37 °C)
  • Laminar flow tissue culture cabinet
  • Flow cytometer - blue laser excitation at 488 nm and proper emission filters.
背景 Natural killer cells represent one of the three major types of lymphocytes (T cells, B cells and NK cells). NK cells play critical role in the innate immune system providing rapid response to protect the body from both tumors and pathogens at the site of infection. They recognize and kill infected or transformed cells, but do not usually harm normal cells. Transmembrane signal transduction generated by interactions between NK cell activating and inhibitory receptors and molecules of ligands which are expressed on the surface of other cell regulate NK cell activation. By recognizing changes or even absence of surface class I MHC molecules, NK cell can distinguish infected and/or stressed cell from normal cell and kill the target cell subsequently. In order to prevent NK cell activation and attack of normal cell, activating signals must be blocked by inhibitory signals. Generally, NK cell activation is regulated by balance between activating and inhibitory signals [1]. Five NK cell subtypes can be recognized in human peripheral blood based on the relative expression of surface markers CD16 (FcγRIIIa, the Fc receptor of immunoglobulin G) and CD56 (NCAM glycoprotein molecule with cell to cell adhesion role) [2], [3], [4]. In combination with other specific surface markers like CD45 (LCA - Leukocyte common antigen) and CD3 (TCR receptor complex component) it is possible to distinguish populations of NK cells from other lymphocytes in human peripheral blood sample. When stimulated with variety of stimuli like interleukin-2, interferon-alpha, mitogens (PWM, PMA), cell antigens (K562, JAR) or viral infections [5], [6] NK cells (CD56+, CD16+, CD45+, CD3- ) express CD69 activation marker (C-type lectin-like receptor) on their surface. Analysis of expression of CD69 marker thus can answer the question what influence particular stimulus has on activation of human natural killer cells.
样本量 100 μL
实验流程 This test is based on the detection of CD69 activation marker on the surface of NK cells after stimulation of human heparinized whole blood with Stimulation Control (PWM) and/or other stimulus. Negative control blood sample, where no stimulating reagent is used, shows no expression of CD69 marker on NK cells. After 24 - 48 h incubation with stimulation reagents the tested blood samples are subjected to the staining with cocktail of monoclonal antibodies labeled with different fluorochromes (anti-CD45 FITC / anti-CD16+CD56 PE / anti-CD3 PE-Cy™5 / anti-CD69 PE-Cy™7) which specifically bind to the antigenic determinants expressed on the surface of leukocytes. The specific staining of blood cells is followed by a lysis of red blood cells. Afterwards, unaffected leukocytes are subjected to analysis by a flow cytometer. The fluorochromes attached to the monoclonal antibodies are excited via laser beam and subsequent emissions of light from the fluorochromes of each cell are collected and analyzed by a flow cytometer. The fluorescence intensity differences enable the separation of cell subsets based on the expression of analyzed antigens.
试剂准备

Preparation of reagents before assay

  1. Aseptically reconstitute lyophilized Stimulation Control using 150 μL of recommended culture medium. Aliquot unused reagent in appropriate volumes, freeze and store for later use at -20 °C. Avoid repeated freeze/thaw cycles.
  2. 10x Lysing Solution is a 10x concentrated and must be diluted 10x with deionized water prior to use (1 volume of concentrated solution with 9 volumes of deionized water). The prepared solution (1x concentrated) is stable for 1 month when stored at room temperature.

样品收集 Use heparinized human whole blood for the examination. The peripheral blood must be processed within 6 hours after sample Collection.
实验流程

For the stimulation of blood samples prepare standard tissue culture test plate and add tested blood into individual wells together with appropriate reagents for negative control sample, positive control sample and for the sample to be stimulated with stimulus of your choice.

  1. Add aseptically into individual wells of tissue culture test plate the following reagents for:
    • negative control sample: 100 μL of culture medium suitable for cultivation of blood samples + 100 μL of blood sample
    • positive control sample: 10 μL of reconstituted Stimulation Control + 90 μL of culture medium suitable for cultivation of blood samples + 100 of μl blood sample.
    • stimulated sample: 10 μL of stimulus of your choice + 90 μL of culture medium suitable for cultivation of blood samples + 100 of μl blood sample.

    • Cultivate the tissue culture test plate with prepared blood samples for 24 - 48 h at 37 °C and 5 % CO2 atmosphere in cell/tissue culture incubator.
    For the examination of blood samples after stimulating step prepare test tubes for negative control sample, positive control sample and for the sample stimulated with stimulus of your choice.
  2. Add 10 μL of Staining Reagent into the tubes destined for:
    • negative control sample
    • positive control sample
    • stimulated sample
  3. Add 50 μL of blood samples from tissue culture test plate wells into test tubes according to corresponding designation. Mix the tubes with a vortex mixer.
  4. Incubate the tubes for 15-20 minutes at room temperature in the dark.
  5. Add 450 μL of diluted lysing solution per test tube. Mix the tube with a vortex mixer.
  6. Incubate for about 5-10 minutes, until the blurry blood sample solution becomes clear.
  7. Analyze the samples immediately using flow cytometer or store the samples at 2-8 °C in the dark and analyze within 24 hours. No further cell fixation is required.

Flow cytometry

Analyze stained samples using flow cytometer equipped with excitation laser 488 nm and proper filters. In order to analyze sufficient number of NK cells (~ 2,000) acquire at least 10,000 - 20,000 leukocytes (CD45+ gating region) per sample. Compensate fluorescent signals prior to or after data Acquisition.

结果分析

Visualize compensated data of negative control sample, positive control sample and stimulated sample in the sidescatter (SSC) versus CD45 FITC dot-plot. Set the gate around lymphocyte population. Visualize CD45+ lymphocytes in a dot-plot CD16+56 PE versus CD3 PE-Cy™5. Separate populations using appropriate gate of natural killer (NK) lymphocytes situated in upper-left quadrant (CD16+56+, CD3-) and T lymphocytes situated in right quadrants (CD3+) on the dot-plot. Finally, plot population of NK cells (CD16+56+, CD3-) as cell count (number of events) versus CD69 PE-Cy™7 in histogram. The data in the histogram for negative control sample are used to generate the discrimination gates defined as negative and positive in the first histogram. It is essential in order to set appropriate gates and calculate the percentage of CD69 positive NK cells (activated cells) in positive control sample and stimulated sample.

限制 仅限研究用
注意事项
  • 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 prolonged exposure of the Staining Reagent to light.
  • Avoid contamination of the reagents.
  • Any non-performance of assay procedure may produce false results.
  • Blood samples are considered as potentially infectious and must be handled with care.
储存条件 4 °C