PBMC Mediated Cytotoxicity Using Celigo Image Cytometry

00:03:29
https://www.youtube.com/watch?v=iqPFiL8-G30

概要

TLDRDr. Leo Jim from Nexalon Bioscience presents an improved method for assessing cell-mediated cytotoxicity assays utilizing image cytometry rather than traditional chromium release assays. The new method employs calcium AM staining for direct counting of live target cells in culture wells, providing a non-toxic, efficient alternative that uses significantly fewer cells and allows for real-time monitoring. The process involves co-culturing PBMC effector cells with K562 target cells and imaging at various time points to observe changes in cytotoxicity, demonstrating better results compared to conventional methods.

収穫

  • 🔬 New method for cytotoxicity measurement using image cytometry
  • 🧬 Image cytometry allows direct counting of live cells
  • ⚠️ Non-toxic alternative to chromium release assays
  • 📊 Enables time course measurement of cytotoxicity
  • 💡 Requires significantly fewer cells compared to traditional methods
  • 👥 Co-culture PBMC effector cells with target cells
  • 📈 Increase in cell-mediated cytotoxicity is observed with higher effector-to-target ratios
  • 🔍 Use of calcium AM staining for live cell identification
  • 🕒 Results show increased cytotoxicity over a four-hour period
  • 📞 Contact Nexalon for more information

タイムライン

  • 00:00:00 - 00:03:29

    Dr. Leo Jim from Nexalon Bioscience introduces a novel method for measuring cell-mediated cytotoxicity using image cytometry, which addresses the limitations of traditional methods like chromium release assays. Unlike hazardous chromium assays that require a significant amount of target cells and only provide endpoint measurements through supernatants, the image cytometry method captures and analyzes live cells directly from wells, allowing for real-time assessments while using twenty times fewer cells. The procedure involves staining K562 target cells with calcium AM, co-culturing them with PBMC effector cells, and imaging over a four-hour span to measure cytotoxicity through cell counting. The results show a direct correlation between PBMC ratios and cytotoxicity, supporting the reliability of this non-toxic and efficient alternative. For further details on this methodology, viewers are encouraged to reach out via Nexalon's website.

マインドマップ

ビデオQ&A

  • What does the new method measure?

    The new method measures cell-mediated cytotoxicity using image cytometry.

  • Why is image cytometry preferred over traditional methods?

    Image cytometry is preferred because it is non-toxic, directly counts cells, allows for time course measurements, and requires significantly fewer cells.

  • What are the steps to perform the assay?

    Collect target cells, stain with calcium AM, co-culture with PBMC effector cells, and image/count live target cells.

  • What does the calcium AM staining do?

    Calcium AM staining allows for the identification and counting of live target cells.

  • How is cytotoxicity assessed?

    Cytotoxicity is assessed by calculating percentage lysis based on cell counts.

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オートスクロール:
  • 00:00:01
    [Music]
  • 00:00:08
    hi I'm doctor Leo Jim from neck salon
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    bioscience today I am presenting a
  • 00:00:13
    improve method to measure your cell
  • 00:00:15
    mediated cytotoxicity assays using image
  • 00:00:18
    cytometry traditionally cell-mediated
  • 00:00:22
    cytotoxicity assays are performed by
  • 00:00:24
    using release assays such as chromium
  • 00:00:27
    LDH or calcium specifically for chromium
  • 00:00:30
    release target cells are labeled to his
  • 00:00:33
    chromium 51 washed and then co-culture
  • 00:00:36
    with effector cells as effector cells
  • 00:00:39
    killed the target cells chromium is
  • 00:00:41
    releasing to the supernal and is
  • 00:00:43
    measured to determine the cell median
  • 00:00:45
    cytotoxicity
  • 00:00:46
    however the problems with this method is
  • 00:00:49
    that it is hazardous and it indirectly
  • 00:00:52
    measures the cells through super named
  • 00:00:54
    it only allows an endpoint measurement
  • 00:00:57
    and requires a significant amount of
  • 00:00:59
    cells in contrast the image cytometry
  • 00:01:03
    method images and analyzes captured
  • 00:01:05
    images directly from the wells using an
  • 00:01:08
    alternative label such as calcium am can
  • 00:01:11
    stain live target cells bright field and
  • 00:01:14
    fluorescent images are acquired and the
  • 00:01:17
    calcium stained live target cells are
  • 00:01:19
    counted at different time points to
  • 00:01:21
    measure cell-mediated cytotoxicity this
  • 00:01:25
    method is non-toxic and directly counts
  • 00:01:28
    cells in each well it also allows for
  • 00:01:30
    time course measurement and uses twenty
  • 00:01:33
    times fewer cells than the release
  • 00:01:35
    assays making it more suitable for
  • 00:01:37
    precious cell types to perform a PBMC
  • 00:01:43
    mediated cytotoxicity assay using k562
  • 00:01:46
    target cells first collect your target
  • 00:01:49
    cells and staying with calcium a.m. then
  • 00:01:52
    see the cells in a 96-well plate second
  • 00:01:55
    add the PBMC effector cells at different
  • 00:01:58
    ET ratios with il-2 third call culture
  • 00:02:02
    to k562 and PBMCs for four hours and
  • 00:02:05
    finally image and count live target
  • 00:02:09
    cells over time using this illegal image
  • 00:02:11
    cytometry the
  • 00:02:15
    cell counts are used to calculate
  • 00:02:16
    percent lysis which showed increasing
  • 00:02:19
    cell-mediated cytotoxicity as ztt ratio
  • 00:02:22
    increased the fluorescent images showed
  • 00:02:25
    the number of calcium positive target
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    cells decrease as ett ratio increased
  • 00:02:30
    with PBMCs the time course results show
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    an increase in cell-mediated
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    cytotoxicity over the four-hour period
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    which is confirming the corresponding
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    fluorescent images in comparison to
  • 00:02:44
    chromium release assay the image
  • 00:02:46
    cytometry method is non-toxic directly
  • 00:02:49
    count cells allows time course
  • 00:02:52
    measurement and uses significantly fewer
  • 00:02:54
    cells there are many peer review
  • 00:02:57
    publications using Sulli go image
  • 00:02:59
    cytometry to perform cell-mediated
  • 00:03:01
    cytotoxicity assays for more information
  • 00:03:05
    on performing cell-mediated cytotoxicity
  • 00:03:07
    assays using image cytometry please
  • 00:03:10
    contact us at NEX alum calm
  • 00:03:17
    you
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    you
タグ
  • cell-mediated cytotoxicity
  • image cytometry
  • calcium AM
  • effector cells
  • PBMCs
  • K562 cells
  • non-toxic
  • real-time monitoring
  • fluorescent imaging
  • cytotoxicity assays