Flowers in Chania

Episode#53 - What are some steps for a successful sort?

Episode#53 - What are some steps for a successful sort?
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    - Welcome to another edition of Ask ExCyte.
    I'm Tim Bushnell with Expert Cytometry,
    and today's question comes from Cliff.
    And Cliff asks: Can you share some tips for your secrets
    for a successful sort?
    First one is go talk to the sort team,
    whoever's gonna be running the sorter,
    to make sure you understand
    how they're running their system, and you can communicate
    information about your sample as well.
    You need to know the rough size of the cell.
    Not flattened down under a microscope slide,
    but the spherical size, because that's gonna dictate
    the nozzle size that you're gonna be able to use
    on your flow cytometer.
    That nozzle size dictates the pressure
    that the system can run at,
    and that therefore dictates
    how fast you can vibrate
    the stream to generate droplets.
    So that information's really critical.
    Second, make sure you filter your samples.
    You do not want to have a clog on the system,
    because that's gonna cause delays for everybody,
    and can result in extensive downtime
    while the clog is cleaned.
    So make sure you filter it right before you go
    to the instrument.
    Make sure you know what type of tube you're collecting into,
    and if you're gonna be doing downstream analysis,
    you probably wanna take those tubes
    and coat them with some sort of protein.
    I typically add my staining buffer and let them sit
    for at least half an hour before I run on this order.
    And last but not least, be careful if you're using trypsin
    to take off adherent cells or breaking down tissue.
    Most people will neutralize trypsin by adding serum,
    but unfortunately when you add serum back, that tends to
    give the cells all they need to start clumping again.
    So you wanna avoid using trypsin.
    I encourage using soybean trypsin inhibitor
    or purified BSA as your neutralizing agent.
    You can also add a little bit of EDTA
    to help eliminate that clumping,
    take away some of those divalent cations.
    I also encourage people to add DNase,
    about 10 units per milliliter.
    This helps to degrade DNA so that the DNA
    doesn't cause the cells to clump.
    But of course, you have to balance the EDTA
    to prevent clumping with the divalent cations
    and the cells' natural processes, with the fact that
    DNase does needs some magnesium to operate.
    So those are some of my steps that I always make sure
    people know about when we're talking about a sort,
    to make sure they're successful.
    Sample prep is so critical, so really focus
    on getting a good, clean sample preparation.
    You may wanna go through the motions
    of preparing your sample, and bring that sample
    to the facility to see
    how well it's gonna run on the machine
    before you actually commit to staining and sorting,
    especially if you're able to get a plentiful sample
    like PBMCs or something of that nature.
    So thanks for your question. If you have any more questions,
    go to getflowtraining.com for more information,
    or send me an email at askexcyte@expertcytometry.
    Thanks for another Ask ExCyte, and until next time,
    stay tuned right here to learn best practices
    in flow cytometry with me, Tim Bushnell,
    and Team ExCyte.
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