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SpermFlowEx® Kit

ABIN3072917 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 SpermFlowEx® Kit is designed for analysis of parameters in human semen using flow cytometry.
品牌 SpermFlowEx®
样品类型 Sperm
产品特性 The SpermFlowEx® Kit is designed for analysis of following parameters in human semen using flow cytometry:
  • Sperm count
  • Leukocyte count
  • Sperm viability
  • Sperm acrosome integrity
  • Presence of intra-acrosomal protein in sperm.

    The analysis should be completed by the examination of sperm motility and morphology using a light microscope in accordance to WHO recommendation.
组件
  • Intra-acrosomal protein FITC
    1 vial containing 0.5 ml of mouse monoclonal antibody against intra-acrosomal protein, FITC labeled.
  • CD45 PE-Cy™5
    vial containing 0.25 ml of mouse monoclonal antibody against CD45, PE-Cy™5 labeled.
  • Fluorescent Count Standard
    1 vials containing 2 ml of fluorescent beads, 1x106 particles/ml
  • Propidium Iodide
    1 vials containing 0.25 ml of propidium iodide solution.
  • Permeabilizing Solution
    1 vials containing 25 ml of the reagent.
试剂未包括
  • Suitable 5 mL test tubes for blood staining (e.g. 12 x 75 mm)
  • PBS buffer
  • Automatic pipettes with disposable tips
  • Vortex mixer
  • Light microscope
  • Centrifuge with rotor suitable for test tubes
  • Flow cytometer - blue laser excitation at 488 nm and proper emission filters.
背景 Lately, infertility of human population is a growing problem. Nearly 20 % of couples suffer from infertility, which is in 1/3 causes attributed to a male factor. Therefore, analysis of semen should play a role in basic non-invasive examination, which could verify or negate andrological cause of infertility. Sperm examination is carried out mainly using the light microscope and obeys the WHO criteria from 2010 [1]. Such examination is subjective and depends on the personal experience of the examiner. When using the flow cytometry the analysis is balanced and objective, since the amount of measured sperms is much greater and detection of cells is provided via specific staining [2]. Main parameters having great impact on the ability of sperm to fertilize are: sperm count, sperm viability, acrosome integrity and presence of an intraacrosomal protein (IAP) [3]. The acrosome contains digestive enzymes (including hyaluronidase and acrosin). These enzymes breakdown the outer membrane of the ovum called the zona pellucida, allowing the haploid nucleus of the sperm penetrate into the ovum. Presence of leukocytes in semen is a mark of an actual inflammation or a venereal disease.[1] WHO laboratory manual for the Examination and processing of human semen. World Health Organization, 5 th edition, 2010 [2] Gilan L, Evans G, Maxwell WM (2005) Flow cytometric evaluation of sperm parameters in relation to fertility potential. Theriogendogy. 63: 445-57 [3] Peknicova J, Chladek D, Hozak P (2005) Monoclonal antibodies against sperm intra-acrosomal antigens as markers for male infertility diagnostics and estimation of spermatogenesis. Am J Reprod Immunol. 53(1): 42-9 [4] Leukocyte Typing IV., Knapp W. et al. (Eds.), Oxford University Press (1989).
说明

  • Sperm count and leukocyte count:
    • Measurement of the sperm count and leukocyte count is based on the addition of an internal standard (fluorescent beads with known concentration) to the semen sample. Detection of leukocytes in the sample is performed by staining with labeled antibody against human CD45 antigen (CD45 PE-Cy™5).
  • Sperm viability:
    • Sperm viability is examined using propidium iodide which permeates through damaged membranes of dead cells and binds to their DNA.
  • Acrosome integrity
    • Measurement of acrosome integrity is based on the detection of an intra-acrosomal protein (IAP), which can be found inside the acrosome. If the sperm acrosome is intact, it is unable to detect IAP. Sperm with damaged membrane has IAP exposed and therefore accessible to the antibody against IAP, hence the protein is detected.
  • Presence of intra-acrosomal protein
    • After permeabilization of sperm membrane, intra-acrosomal protein is exposed to the antibody against intra-acosomal protein (IAP) and thus is detected. In case, that sperm does not contain intra-acrosomal protein (IAP), the protein is not detected after permeabilization.

样本量 50 μL
样品制备

Treat semen samples within 8 hours after ejaculation. Store semen samples at room temperature.

实验流程

Check the sperm count using a light microscope. Dilute the semen 1:1 or 1:9 with PBS according to the sperm count (the greater the count the greater the dilution).

Sperm and leukocyte count

  1. Pipette 50 μL of diluted semen to a sample tube. Add 10 μL of CD45 PE-Cy™5 reagent.
  2. Mix the sample and incubate for 20 minutes at room temperature.
  3. Add 50 μL of Fluorescent Count Standard (first drop well mixed standard in an empty microtube and then pipette the exact volume of the standard to the sperm sample).
  4. Add 0.5 mL of PBS, mix the sample and analyze using a flow cytometer.

Sperm viability ol>
  • Pipette 50 μL of diluted semen to a sample tube.
  • Add 10 μL of Propidium Iodide solution.
  • Mix the sample and incubate for 20 minutes at room temperature.
  • Add 0.5 mL of PBS, mix the sample and analyze using a flow cytometer.

  • Acrosome integrity
    1. Pipette 50 μL of diluted semen to a sample tube.
    2. Add 1 mL of PBS.
    3. Mix well and centrifuge for 5 minutes at 150 g.
    4. Wash the pellet twice (repeat the 2nd and 3rd step).
    5. Add 10 μL of Intra-acrosomal protein FITC antibody to the pellet.
    6. Mix the sample and incubate for 20 minutes at room temperature.
    7. Add 0.5 mL of PBS, mix well and analyze using a flow cytometer.

    Presence of intra-acrosomal protein
    1. Pipette 50 μL of diluted semen to a sample tube.
    2. Add 1 mL of Permeabilizing Solution which was allowed to warm up to room temperature (20-25 °C).
    3. Mix the sample and incubate for 15 minutes at room temperature.
    4. Centrifuge for 5 minutes at 150 g.
    5. Add 1 mL of PBS to the pellet, mix and centrifuge for 5 minutes at 150 g.
    6. Wash the pellet twice (repeat the 5th step).
    7. Add 10 μL of Intra-acrosomal protein FITC antibody to the pellet.
    8. Mix well and incubate for 20 minutes at room temperature.
    9. Add 0.5 mL of PBS, mix well and analyze using a flow cytometer.

    Flow Cytometry

    Analyze stained samples using flow cytometer equipped with a 488 nm excitation laser and appropriate filter set-up. Fluorescent Count Standard is detected in FITC fluorescence detector as well as monoclonal antibody against intra-acrosomal protein. Fluorescence of propidium iodide and PE-Cy™5 label is detected in PC5 or PerCP fluorescence detector.

    结果分析

    Sperm and leukocyte count:
    Visualize measured data as a dot-plot, where FITC fluorescence intensity is on the X-axis and forward-scatter (FSC) is on the Y-axis. Set gates: gate A for all cells, gate B for sperms and gate C for Fluorescent Count Standard (fluorescent beads). Then visualize events from the gate A as a dot-plot, where the X-axis represents fluorescence intensity of PE-Cy™5 dye and the Y-axis represents side-scatter (SSC). On the contrary to the sperms, leukocytes are CD45 positive. Separate the bright population of leukocytes using gate D. Calculate sperm and leukocyte count using following formulas:

    S = (gateB/gateC) x dilution [106 sperm/mL]
    L = (gateD/gateC) x dilution [106 leukocytes/mL]

    S represents sperm count in ejaculate L represents leukocyte count in ejaculate Gate B represents event count in gate B Gate C represents event count in gate C Gate D represents event count in gate D

    Sperm viability:
    Visualize measured data as a forward-scatter (FSC) versus side-scatter (SSC) dot-plot. Set the gate around sperm cells. Then visualize sperm cells in a histogram , where the X-axis represents fluorescence intensity of propidium iodide in PE-Cy5 or PerCP channel. Separate positive and negative sperm cells using appropriate gate. Negative population represents viable sperms, while PI-positive population represents non-viable sperms. Viability of sperms is represented by the percentage of viable sperms from all sperm cells.

    Acrosome integrity and presence of intra-acrosomal protein:
    Visualize permeabilized and non-permeabilized samples stained using antibody against intra-acrosomal protein as a forward-scatter (FSC) versus side-scatter (SSC) dot-plot and set the gate around sperm events. Then visualize sperm events in a histogram, where the X-axis represents fluorescence intensity in FITC fluorescence detector. Separate positive and negative sperms using appropriate gates.

    Expected values:
    Normal values according to WHO [1]:

    • Sperm count in semen > 15x106 /mL
    • Leukocyte count in semen < 1x106 /mL
    • Sperm viability >58 %
    Normal acrosomal values:
    • Acrosome integrity of sperms <30 % pathological
    • Presence of intra-acrosomal protein >90 % sperms

    限制 仅限研究用
    储存条件 4 °C