PBMC Mediated Cytotoxicity Using Celigo Image Cytometry

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

Ringkasan

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.

Takeaways

  • 🔬 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

Garis waktu

  • 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.

Peta Pikiran

Video Tanya Jawab

  • 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.

Lihat lebih banyak ringkasan video

Dapatkan akses instan ke ringkasan video YouTube gratis yang didukung oleh AI!
Teks
en
Gulir Otomatis:
  • 00:00:01
    [Music]
  • 00:00:08
    hi I'm doctor Leo Jim from neck salon
  • 00:00:11
    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
  • 00:02:27
    cells decrease as ett ratio increased
  • 00:02:30
    with PBMCs the time course results show
  • 00:02:35
    an increase in cell-mediated
  • 00:02:36
    cytotoxicity over the four-hour period
  • 00:02:38
    which is confirming the corresponding
  • 00:02:41
    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
  • 00:03:26
    you
Tags
  • cell-mediated cytotoxicity
  • image cytometry
  • calcium AM
  • effector cells
  • PBMCs
  • K562 cells
  • non-toxic
  • real-time monitoring
  • fluorescent imaging
  • cytotoxicity assays