Flow Cytometry Shared Resource

Transcript

• 00:00Hi. My name is Anne
• 00:02Haberman. I'm the scientific director
• 00:04of the flow cytometry facility.
• 00:06I co direct with Leslie
• 00:08Devine who's, also attending this
• 00:11presentation today.
• 00:15As
• 00:16most of you know, flow
• 00:18cytometry is a technique that,
• 00:21allows every object to be
• 00:23interrogated for fluorescence and light
• 00:25scattering properties, including,
• 00:27some that are non fluorescent.
• 00:29Using this technique, the marker
• 00:31expression
• 00:33of, many, protonacious
• 00:35antigens typically detected with fluorescently
• 00:37conjugated antibodies
• 00:39can be assessed and very
• 00:40precisely,
• 00:42quantified for statistics.
• 00:46This is not showing correctly.
• 00:48Sorry.
• 00:50So, shown here is just
• 00:52some very simple gating of,
• 00:54just a small number of
• 00:55parameters from, twenty four color
• 00:58panel,
• 00:58that is easily obtained on,
• 01:01a number of our,
• 01:03analytic flow cytometers.
• 01:07In addition to analysis, fluorescently
• 01:09tagged cells can also be
• 01:10sorted and collected for downstream
• 01:12applications,
• 01:13such as cell culture assays,
• 01:15DNA or RNA seek, including
• 01:17single cell RNA seek,
• 01:19adoptive cell transfers for subsequent
• 01:21in vivo studies.
• 01:24Really, anything that you can
• 01:25imagine in which you would
• 01:26need
• 01:27purified,
• 01:28cells with great accuracy
• 01:30on its,
• 01:31protein expression.
• 01:34Cells can be collected into,
• 01:36a variety of different types
• 01:37of tubes
• 01:38or index sorted into plates
• 01:41based on,
• 01:42intensity of expression of your
• 01:44markers of interest.
• 01:46And this latter technique can
• 01:47be helpful for a post
• 01:49culture retrospective
• 01:50analysis
• 01:51of an in vitro assay,
• 01:53say for example.
• 01:56Here's the list of equipment,
• 01:58that is distributed
• 01:59across campus.
• 02:01In addition,
• 02:03to,
• 02:04traditional,
• 02:05analytic flow cytometers and cell
• 02:07sorters that I'll describe more
• 02:09in a minute,
• 02:10we also,
• 02:12can support the use of,
• 02:14imaging flow cytometry on an
• 02:16AMNES image stream,
• 02:17flow cytometer,
• 02:19and,
• 02:20also support Luminex style assays.
• 02:23We're providing service to really,
• 02:26a large number of labs
• 02:28in nearly every department on
• 02:29the Yale School of Medicine
• 02:31campus.
• 02:33And, having been around for
• 02:35quite a while since, two
• 02:36thousand seven, we have really
• 02:38broad experience supporting cytometry of
• 02:40all types of samples and
• 02:42really every
• 02:43object or or cell size,
• 02:45including the very small, like,
• 02:46bacteria,
• 02:48or, extracellular
• 02:49vesicles,
• 02:50to the very large.
• 02:54We have support for both
• 02:55the independent operation of instruments
• 02:58or a fee for service
• 02:59options with advanced notice.
• 03:01And I'd like to mention,
• 03:03YCC members receive
• 03:05a ten percent co pay
• 03:06on our fees,
• 03:08which are, a net zero
• 03:10cost recovery,
• 03:12scheduling approach.
• 03:14The instruments are distributed across
• 03:16campus.
• 03:18We have two major hubs,
• 03:19and that would be in
• 03:20three hundred George Street and
• 03:22also in the TAC building,
• 03:24as well as a self
• 03:25sorter inside the biosafety level
• 03:27three facility in LEPH,
• 03:29and a sorter and, an
• 03:31analytic,
• 03:32Fortessa
• 03:33in the Amistad building.
• 03:38We have, quite a few
• 03:39staff who are available to
• 03:41guide, train, or assist you
• 03:43really in all elements
• 03:45of data acquisition,
• 03:47or analysis.
• 03:49You may have some very
• 03:51fundamental,
• 03:52de novo level,
• 03:53questions,
• 03:55that that we can, assist
• 03:56you with,
• 03:57all the way to very
• 03:59high parameter,
• 04:01clustering approaches similar to the
• 04:03t SNE analysis that you
• 04:04might do with single cell
• 04:06RNA seq.
• 04:08Some of our staff is
• 04:09devoted to just the support
• 04:11of the analytic flow cytometers.
• 04:14But the vast majority of
• 04:15our staff
• 04:16is very cross trained and
• 04:18may primarily spend their time,
• 04:21assisting with self sorting at
• 04:22the instrument,
• 04:24but are are aware of
• 04:25and, and can,
• 04:28help to guide you,
• 04:30to choose
• 04:31the correct analytic instrument that
• 04:33might pair best with their,
• 04:36cell sorter or might, better
• 04:38address your biological questions.
• 04:43Our analytics cytometers,
• 04:45are available for use for
• 04:46independent use, and that's primarily
• 04:48how they are used.
• 04:50Broadly kind of fall into
• 04:51two categories.
• 04:53Those that separate light in
• 04:55a conventional,
• 04:57fashion,
• 04:58and that would be a
• 04:59lot of instruments manufactured by
• 05:01BD,
• 05:02and including their numerous symphonies
• 05:05for TESAs, some ELSA twos.
• 05:07And the CytoFlex,
• 05:10flow cytometer is very sensitive
• 05:12with avalanche detectors and manufactured
• 05:15by Beckman Coulter.
• 05:17We also,
• 05:18have two auroras
• 05:20that use, instead a different
• 05:22approach,
• 05:24that, would be referred to
• 05:25as spectral detection.
• 05:27So, whereas with conventional detection,
• 05:29light is parsed out a
• 05:31little more discreetly,
• 05:33With spectral detection instead,
• 05:35all the light, being emitted
• 05:37off of each of the
• 05:38lasers is collected.
• 05:40And these use, slightly different
• 05:42approaches to distinguishing one of
• 05:44four four four another, but
• 05:46this latter
• 05:47one is more compatible
• 05:49with very large numbers of,
• 05:51of fluorophores being used at
• 05:53the same time.
• 05:54So with the symphony, the
• 05:56Bigfoot, or the Wura, twenty
• 05:57plus colors are easily distinguished.
• 06:00With the aurora,
• 06:03many more could theoretically be
• 06:05detected. And and, and these
• 06:07days, the limit is much
• 06:07more about what you can
• 06:09find commercially pre prepared in
• 06:11the way of distinguishable fluorophores,
• 06:13less so what the instrument
• 06:15can,
• 06:16can
• 06:17distinguish itself.
• 06:20I had mentioned the AMNOS
• 06:22Image Stream Imaging Flow Cytometer.
• 06:24This instrument can acquire images
• 06:26of cells that are in
• 06:28suspension.
• 06:29So whereas previously,
• 06:32you might have had to
• 06:33have used, confocal to take
• 06:35images of large numbers of
• 06:37cells on in in order
• 06:38to,
• 06:39then, perform your own image
• 06:41analysis for that quantification of
• 06:43location
• 06:44of the fluorescence within a
• 06:46cell.
• 06:48Now you can make a
• 06:48single cell suspension,
• 06:50and with up to, eight
• 06:52colors now,
• 06:54be able to perform studies
• 06:56that would help you
• 06:57instead distinguish,
• 06:58say
• 06:59surface versus internalized components,
• 07:03refine your cell cycle, mitosis
• 07:05or DNA damage studies,
• 07:08autophagy, cell signaling, cell cell
• 07:10interactions,
• 07:11you know, such as TB
• 07:12interactions,
• 07:13defining the synapse, quantifying what's
• 07:16at the synapse,
• 07:17asymmetric cell division, say, for
• 07:19example.
• 07:20All these things are, possibilities,
• 07:22that can be a real
• 07:24slam dunk,
• 07:25for,
• 07:26other orthogonal
• 07:28assays for a higher impact
• 07:29paper.
• 07:30There's staff support for this,
• 07:32as is everything in the
• 07:33Flow Core,
• 07:34for both the data acquisition
• 07:36and the analysis.
• 07:38We can help to advise
• 07:39you on,
• 07:41what colors would be appropriate
• 07:43for where this elements of
• 07:44sample preparation.
• 07:46There's very intuitive image analysis,
• 07:50including what will soon arrive,
• 07:52a more AI driven wizard
• 07:54if,
• 07:56if that's what you would
• 07:57prefer.
• 07:58That also can incorporate traditional
• 08:00flow cytometry gating
• 08:03as well as applying
• 08:05image
• 08:06masks and features
• 08:07that can also be gated
• 08:09on plots.
• 08:11So I don't really have
• 08:12time to delve much more
• 08:14into this
• 08:15but this is a powerful
• 08:17instrument.
• 08:19And lastly, I'd like to
• 08:21describe other lines of service
• 08:23that are a little more
• 08:25wet bench oriented.
• 08:26Let's say for example with
• 08:28the Luminex,
• 08:30we can provide sample preparation,
• 08:34data acquisition
• 08:35or analysis or train you
• 08:37to do these things yourself
• 08:38on this instrument.
• 08:41Along these lines of, what
• 08:43had previously been referred to
• 08:44as immune monitoring services
• 08:46includes cryopreservation
• 08:48of human serum or peripheral
• 08:50blood,
• 08:52of human blood,
• 08:53staining of samples for flow
• 08:55cytometry.
• 08:56We can run those same
• 08:57samples for you on a
• 08:58flow cytometer and do the
• 09:00analysis or any fragment
• 09:02subsection of that.
• 09:04It's possible to perform same
• 09:06day cell sorting of human
• 09:08samples, but typically this is
• 09:09restricted to small sample sizes
• 09:12and a short time commitment,
• 09:14or can be accommodated
• 09:16after hours.
• 09:18And, finally, I'd like to
• 09:19mention that we had validated
• 09:21a number of high parameter
• 09:23antibody panels,
• 09:25for those of you who
• 09:25don't wish to design your
• 09:27own. And and at the
• 09:28moment, these are purchasable.
• 09:31And I will stop there
• 09:33and answer any questions.
• 09:37Thank you.
• 09:44K.
• 09:45We will move on to
• 09:47our last presentation.
• 09:50Hello, everybody. My name is
• 09:51Kamala Krambaipudi.
• 09:53I manage the Irvator share
• 09:55resource, resource center here. We're
• 09:57part of the cancer center
• 09:58support,
• 09:59grant share resources.
• 10:03ISR is supervised by doctor
• 10:05Stern,
• 10:06and reviewed annually by the
• 10:07YCC shared, oversight committee.
• 10:12The shared resource is managed
• 10:13by doc director doctor Jay
• 10:15Chen.
• 10:17He is the, director for
• 10:18ISR. He's a professor of
• 10:20therapeutic radiology, member of the
• 10:21Yale Cancer Center, and vice
• 10:23chair for the physics research
• 10:25and education in therapeutic radiology.
• 10:27He has about three years
• 10:28of experience in radiation physics,
• 10:30and then he's an
• 10:31in house expert on gamma
• 10:33and x rays.
• 10:37He's also a fellow of
• 10:38the American Association of Physicists,
• 10:40in medicine and the chief
• 10:41physicist of Smile Against Hospital
• 10:43at Yale
• 10:44New Haven Hospital.
• 10:46So our mission of the
• 10:48shared resource irradiated
• 10:50shared resource is to provide
• 10:51easy and reliable access to
• 10:52cutting edge gamma and x-ray
• 10:54irradiation technologies,
• 10:55services, and scientific consultations
• 10:57for all the cancer researchers
• 10:59at Yale.
• 11:01So we have four objectives
• 11:03and aims. Aim one being
• 11:05providing convenient and reliable access
• 11:07to state of the art
• 11:08gamma ray and
• 11:09global digit, x-ray radiators with
• 11:11cost effective radiation for all
• 11:13YCC members.
• 11:15We provide reliable access to
• 11:16all the radiators. We offer
• 11:17expert help with experimental design.
• 11:20We train all our users
• 11:21to operate the machines safely
• 11:23and keeping in line with
• 11:24EHS protocols,
• 11:26and we keep make sure
• 11:26we keep the machines running
• 11:27twenty four by seven.
• 11:32The key services of ISR,
• 11:34we provide radiation physics and
• 11:35technical consultation on the best
• 11:37use of all the radiators.
• 11:38We provide periodic quality assurance
• 11:41test to ensure accurate dose
• 11:42deliveries delivered by all radiators.
• 11:44We train our users and
• 11:45help with compliance.
• 11:47We also escort users if
• 11:49they use a gamma radiator,
• 11:51then if they don't wanna
• 11:52go any background checks, which
• 11:53I'll talk about later.
• 11:55We also make sure we
• 11:56maintain our latest all year
• 11:57on.
• 11:58We do QH every month,
• 12:00every week, quarterly, and annually.
• 12:05So, we have, different equipment.
• 12:08We have a CCM gamma
• 12:10ray is related.
• 12:11We have four precision x
• 12:13vat three twenty kilowatts irradiators
• 12:14across campus.
• 12:16We have our newest addition,
• 12:17the SARP, the small animal
• 12:19radiation research platform, image guided
• 12:21accelerator,
• 12:22and we also have radiation
• 12:23proximity equipment.
• 12:25Briefly,
• 12:26CGM137
• 12:27Gamma radiator provides,
• 12:29high penetration gamma rays,
• 12:31for whole body or cell
• 12:32variations.
• 12:33The Precision x y three
• 12:35twenty's, they deliver tunable kV
• 12:36x rays for cell tissue
• 12:38and small animal radiation with
• 12:40controlled dose rates. The SARP,
• 12:42enables a CT guided, highly
• 12:44targeted radiation for precise small
• 12:46animal research.
• 12:48We also have radiation dosimeter
• 12:49equipment, which support both cell
• 12:51and animal studies.
• 12:53So, this,
• 12:55this is how a gamma
• 12:56radiator looks like.
• 12:58It delivers up to six
• 13:00six and then six hundred
• 13:01sixty two kV gamma rays.
• 13:02It supports molecular samples, cell
• 13:04cancers, tumor cells, and small
• 13:06animals.
• 13:07It use it's also used
• 13:08for wide dose rate range
• 13:10and flexible collimation, and it
• 13:11has been a core tool
• 13:12for many years since nineteen
• 13:14ninety for all many YCC
• 13:15studies.
• 13:17We will be, removing this
• 13:18machine in the next year
• 13:19as part of a chip
• 13:20program
• 13:21to play and replacing this
• 13:22with a new X-ray retail.
• 13:26So we have a four
• 13:28X-ray machines across campus.
• 13:31These are X-ray three twenties,
• 13:33made by precision.
• 13:34They're used for rating cell
• 13:35cultures, beta layers, bacteria, insects,
• 13:37and small and small animals
• 13:38like rodents, mice, and rats.
• 13:40It's supposed both targeted and
• 13:42whole body radiation,
• 13:43used for studies in tumor
• 13:45and normal tissue response,
• 13:47hematopoietic depletion, immunotherapy relation with
• 13:49imaging research.
• 13:50The output contain, it goes
• 13:52up to three grades per
• 13:53minute at fifty centimeters SSD,
• 13:55distributed across four medical campus
• 13:57buildings for community access.
• 13:59So we have four x-ray
• 14:00machines, one located in TAC,
• 14:02one located in Amistad, one
• 14:03located in Josh, and another
• 14:05one located in LCI building.
• 14:07And this is a gamma
• 14:08ray radar.
• 14:10So our newest edition is
• 14:11called the SARP, small animal
• 14:13radiation research platform.
• 14:14It's an advanced multifunctional accelerator
• 14:17designed specifically for animal research.
• 14:19So what it is is
• 14:20a two twenty five kilowatt
• 14:21accelerator designed specifically for high
• 14:23precision irradiation of mice and
• 14:25rats.
• 14:26The key features of this
• 14:27machine would be cone beam
• 14:28CT imaging,
• 14:29a three d treatment planning
• 14:31just like a miniature clinical
• 14:32relation therapy planning system. It
• 14:34has a movable gantry with
• 14:35a robotic stage so beams
• 14:37can come from many angles
• 14:38for precise targeting.
• 14:40It also has high sorry
• 14:42it has a high reproducibility.
• 14:46So why it matters, it
• 14:47it allows focal aggregation of
• 14:49instead of whole body exposure
• 14:50so you can target precisely
• 14:52or what organ you wanna
• 14:53treat with.
• 14:54It ex reduces normal tissue
• 14:56injury by sparing healthy areas.
• 14:58It's essential for studying tumor
• 14:59response, immunology, DNA repair, and
• 15:01normal tissue toxicity.
• 15:03So in a simple summary,
• 15:04it's a clinical reason that
• 15:05we devised, but scaled down
• 15:07for mice.
• 15:09So this is how it
• 15:10looks like.
• 15:14So the mouse goes on
• 15:15the bed over here. There's
• 15:17a CT detector. The camera
• 15:19takes a three d convinity
• 15:20image.
• 15:21Your X-ray head is on
• 15:22the top.
• 15:23We have a collimator.
• 15:25You can choose your own.
• 15:26We have we have five
• 15:27different collimators of various tumor
• 15:29sizes. You can choose your
• 15:30collimator and,
• 15:32treat them, through the mouse
• 15:33or the rat with whatever
• 15:35tumor it has. So this
• 15:36is a treatment time software.
• 15:37Here, like, I show you
• 15:38a lung tumor
• 15:40in the lung of a
• 15:41mouse so you can target
• 15:43just the tumor in the
• 15:44mouse.
• 15:47So we also have radiation
• 15:48dosimeter equipment. We have INGMO
• 15:50based absolute dosimetry.
• 15:52It measures absolute dose and
• 15:54it converts collected charge into
• 15:56and converts that into a
• 15:57citric rate. It's used to
• 15:58calibrate our machines and we
• 16:00use it for daily and
• 16:01monthly
• 16:02QAs.
• 16:03It's sensitive to set up
• 16:04an environmental corrections.
• 16:06We also have a EBT4
• 16:07filament dosimetry. It provides two
• 16:09d dose distribution. It's great
• 16:11for checking uniformity, beam profiles,
• 16:13and small fields.
• 16:14It requires scanning and calibration
• 16:16curve. It's good for relative
• 16:18dose but not for the
• 16:18absolute dose.
• 16:20We also have a TLD
• 16:21dosimeter system.
• 16:23It's useful where films or
• 16:25cham ion chambers or gaphomic
• 16:27films, EBT four films can
• 16:29go, and it's
• 16:31measures a point dose. It's
• 16:32good for verification of delivered
• 16:33dose. We also have a
• 16:35free chemical dosimeter.
• 16:37It measures relation dose, but
• 16:38how much how much it
• 16:39oxidizes ferrous ions in a
• 16:41specific sulfuric acid solution.
• 16:44So some of our experimental
• 16:46setups on gamma and actuators,
• 16:48we have a whole mouse
• 16:49pie, for mouse whole body
• 16:50radiation.
• 16:51We have shields available built
• 16:53for a brain radiation or
• 16:54a flank tumor radiation.
• 16:56You can always do tissue
• 16:58culture plates and culture flask
• 17:00for cell radiation.
• 17:02We also have two years
• 17:03filters for harder beams and
• 17:05aluminum two million millimeter filter
• 17:06for a softer beam,
• 17:08so we can, always use
• 17:10those filters too.
• 17:14So, trading and access
• 17:16is basically very simple. You
• 17:17have to do three three
• 17:18step three steps, get an
• 17:19online training with EHS,
• 17:20obtain a validation to submit
• 17:22it, fill out a form,
• 17:22and submit to EHS, and
• 17:24hands on training with me,
• 17:25and you would be able
• 17:26to use,
• 17:27any of the exclamations across
• 17:28campus.
• 17:32So same thing. Companies require
• 17:33acts required training and a
• 17:35plain relation to submit it.
• 17:36For a common radiator, you
• 17:37need to submit a background
• 17:38clearance form with with your
• 17:40weight with EHS. It takes
• 17:41about a while, but you
• 17:43don't have to wait for
• 17:44the background clearance. I can
• 17:45export escort you to the
• 17:46common radiator and perform your
• 17:47experiments for you.
• 17:51So all our scheduling and
• 17:52training is done via PPMS.
• 17:54I'll provide the link up
• 17:55here.
• 17:56Please access this link, and
• 17:57everything's up. All all the
• 17:59information we need for training
• 18:00and scheduling is up on
• 18:01the PPMS website.
• 18:04So quickly,
• 18:05some of our research,
• 18:07here, we have a lung
• 18:08cancer presentation with doctor Hayman
• 18:10and doctor Contessa.
• 18:11So we use he they
• 18:12use the x-ray three twenty
• 18:13to deliver fractional doses for
• 18:14cell and most studies.
• 18:16We had doctor Ryan Jensen
• 18:17for DNA repair. He used
• 18:19DNA extra three twenty to
• 18:20induce DNA breaks to measure
• 18:22the RAD fifty one foci.
• 18:24We also have a humanized
• 18:25skeletal model. We talked to
• 18:26Flavwell's lab. They use the
• 18:28sublethal x-ray conditioning
• 18:30for enable HSC engraftment in
• 18:32mice.
• 18:33And we also have doctor
• 18:34Pillai,
• 18:34from George Billing. They use
• 18:36the X-ray three twenty to
• 18:37induce DNA damage to test
• 18:38synthetic lethal responses.
• 18:41So,
• 18:42for the fiscal year of
• 18:43twenty twenty five,
• 18:45we had about fifty two
• 18:46PIs and twenty one departments
• 18:47that use our machines. Ninety
• 18:49Ninety eight percent of our
• 18:49members are YCC members.
• 18:51So, we we had a
• 18:53use of a thousand two
• 18:54two hundred for the fiscal
• 18:55year twenty twenty five.
• 18:58Quickly about the service rates,
• 18:59we charge about hundred and
• 19:00thirty dollars per thirty minutes.
• 19:02With assisted, that mean I
• 19:03would be doing separation for
• 19:04you. If you get trained
• 19:06and use it by yourself,
• 19:06it's sixty five dollars for
• 19:07thirty minutes. You don't need
• 19:09a you need just need
• 19:09a training, and you have
• 19:10an expert access, meaning you
• 19:11can access the machine anytime
• 19:13area of the week.
• 19:16So this is our contact
• 19:17information here, doctor Jay Chen,
• 19:18and that's me,
• 19:20And our website and people
• 19:21in the website isn't included
• 19:22in the presentation over here,
• 19:24which I'll share with you.
• 19:25Thank you, everybody. Any questions?
• 19:29Thank you so much.
• 19:33Do we have any questions,
• 19:35for this or any of
• 19:37the other shared resources?
• 19:46Alright.
• 19:47Well,
• 19:48before
• 19:49everyone just hold tight. Tanya's
• 19:51gonna give the information about
• 19:52the survey. I just wanted
• 19:53to thank,
• 19:54all of the directors of
• 19:55the shared resources for their
• 19:56time today for giving this
• 19:58very quick introduction.
• 20:00And, again, survey is really
• 20:01important because this is gonna
• 20:03tell us what our next
• 20:04steps are, and what our
• 20:06asks are. So please just
• 20:07take the time to do
• 20:08that.
• 20:12Okay. So as you guys
• 20:14heard,
• 20:15we will be sending a
• 20:16post event survey.
• 20:18Please, please, please,
• 20:20take it's about two minutes.
• 20:22So please take the time
• 20:23to complete it. All responses
• 20:25will
• 20:26be fully anonymous.
• 20:27However, there is an option
• 20:29to put your name in
• 20:31a different survey,
• 20:32if you like to be
• 20:33entered into a raffle, and
• 20:35we will be selecting two
• 20:36winners.
• 20:37So thank you again,
• 20:39to everyone and to all
• 20:40the shared resources leaders for
• 20:42joining us. I hope everyone
• 20:43has a great afternoon.
• 20:45Thank you.
• 20:47Bye. Bye. Thank you.
• 20:51Thank
• 20:53you. Alright.