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hello everybody and welcome to today's.webinar entitled multicolor panel.building in flow cytometry a helpful.guide to optimizing panel building in.flow cytometry your host will be.Sebastian headland one of our flow.cytometry specialists here at bio-rad.this is the fourth in our series of flow.cytometry webinars with more to follow.Sebastian obtained his degree from the.Institute of Technology link shopping.University Sweden followed by an MSC in.medical biology from the Faculty of.Health Sciences from the same University.he then moved to Freiburg Germany where.he obtained his PhD in immunology and.molecular biology at the Max Planck.Institute of immuno biology and.epigenetics the focus of his thesis was.the role of micro RNA 1:55 in innate.immunity and immunopathology he then.left academia in the beginning of 2013.and started to work as a dedicated flow.cytometry specialist before joining.buyer ad in 2016 as a flow cytometry.specialist for Scandinavia Eastern.Europe Israel and South Africa as we're.trying to make this webinar as.interactive as possible please submit.questions or comments via the Q&A box.located at the bottom of the screen.throughout the webinar we will try to.answer as many questions as possible in.this session following the main.presentation should any questions remain.unanswered we will reply by email the.webinar will be recorded and an.on-demand version will be available.shortly after the webinar on the bio-rad.antibodies website should you encounter.any technical problems during the.webinar please click on the question.mark at the bottom of the screen this.will bring up the technical guide which.describes solutions for the most common.technical problems in addition to.webinars we regularly post blog articles.not just on flow cytometry but on other.applications and research areas we also.have blog posts on current scientific.discussions and the latest biomedical.discovery.please feel free to check out the blog.and comment on one of the many articles.in addition you can follow us on social.media to stay up to date with any.antibody resources news and products our.social media channels can be accessed.via the icons at the bottom of the.screen during the webinar you can.interact with us on Twitter by.submitting questions or webinar feedback.using the hashtag bio-rad webinars I.hope you will enjoy the webinar and now.handing over to you Sebastian.thanks for that introduction and hello.everyone first off I would really like.to acknowledge my colleague Sharon.Sanderson from bioriod antibodies who.done all the practical work for this.panel design webinar her help has been.really invaluable and I could not have.done this webinar presentation without.her assistance the focus of today's.webinar will be panel design and we were.looking to the different parameters that.play a crucial role for the success of.your panels with the final aim that you.hopefully feel that you have gained a.better understanding on how to build and.improve your own multicolor panels the.agenda for this webinar will be.instrument fluorophore availability.floor for brightness antigen density and.spectral overlap the cornerstones of.tank design before doing anything else.it's fundamental to know which flow.cytometry you have available and what.configuration lasers filters detectors.it is simply a waste of time to design a.panel if you cannot run it later so.always check state checks the instrument.first in this webinar we will use the.novel by rad 35 Senate cell analyzer and.it carries up to five lasers including.the 355 nanometer UV laser the 405.violet laser 488 blue laser 561 yellow.green laser and the 640 red laser it.also has 30 parameters of which 27 are.dedicated fluorescent detectors it is.also equipped with an optical filter.check called a 35 I that verifies that.the correct filters are installed so.that no filter mistake can ruin your.experiments.top of this 35 can acquire up to a.hundred thousand events per second and.always comes with a flexible and high.throughput loader allowing you to run.samples in anything from a single tube.all the way up to 384 well plates so all.in all it's a flexible flow cytometer.with great potentials multicolor flow.cytometry.for more information go on online to the.city 5 webpage ww-why red commie 527.colors will get us a long way and at the.glance we can see that there is five.detectors on the UV laser line seven.colors on the violet laser line four.colors on the blue laser seven colors on.the yellow-green and four colors on the.on the red so it's quite easy to make.the conclusion that our flora four.choices for this panel design are not.going to be limited by the 35 so after.establishing that the instrument.configuration will not give us any.initial limitations to floor four.choices we will move on to look at the.flora for availability in order to.design a panel we first need to know.what we want to study in this webinar.we'll study natural killer cells and.natural killer T cells in whole blood.from human with those two cell.populations there are several subsets.and it is some of them that we aim to.identify in the panel that we're showing.you today in order to do this we need.five different markers CD 3 CD 8 CD 16.CD 56 and CD 57 keep in mind that out of.a flow cytometry perspective we do not.need to know the function of these.markers the only thing we need to know.is on which cells they are expressed for.me I always like to make a mind map as.shown here on the slide to get an.overview of which markers that are.expressed on which cells obviously we.could use even more markers for this.analysis as well but to prove to prove.the principles of panel design.disestablished markers will be perfect.and even if you want to design bigger.panels the same rules will still apply.and in this case we will be looking for.two subsets of NK cells both of them are.CD 56 positive the one subset is then.positive for feeding CD 57 while the.other one is CD 57 negative but CD 16.positive also we are trying to identify.six subsets of n key T cells where the.starting point is that all of them are.positive for CD three and CD 56 and.after this is established we are then.looking for three different populations.that we are identifying with by three.intensity of CD eight plus that they are.either CD 57 positive or negative so all.in all six subsets of MKT cells.me personally I do not come from an NK.NK t background but from what I know.from these cells do not only play a.crucial role in immuno surveillance but.they display cytotoxic activities.against transformed or viral infected.cells and are also important an.important source of chemicals and.cytokines highly impacting on adaptive.immune responses that is why these cells.hold a lot of interest in for instance.cancer therapies Diagnostics and.translational medicine that is why it.might be beneficial to try and design a.panel that can be used for these.applications but before getting ahead of.ourselves and just dive directly into.designing our panel it's crucial to see.in which fir first these markers are.available it is really annoying not to.speak of frustrating to build a perfect.theoretical panel only to find that some.of the antibodies cannot be found.conjugated with certain fur offers so to.avoid this always make it a habit to.have a look at which floors you have at.your disposal already from start keep in.mind if you do not find your favorite.antibody conjugated to the fur for your.needs there is also several conjugation.kits available so you can do it yourself.and we have the ready link antibody.conjugation kits for small amounts of.antibodies and the links kits for larger.amounts of antibodies so it's easy to.use and result in conjugated antibodies.in just over an hour.in some cases it is also possible to ask.the manufacturer to make these specific.antibodies for you if we look at the.first example of panel design we will.use a method that I think many of you.can relate to we will just use what is.available or what we have in the fridge.this method is kind of like playing on.the lottery and probably the likelihood.of getting a good panel is about this.likely as winning the jackpot in this.example we had the following antibodies.available CD 56 conjugated to Percy.piece i 5.5 CD 3 with te t d 8 with PE.Alexa for 750.it is 16 with Alexa 4 647 and CD 57 with.APC sy 7 a nice feature with every.software on the 35 is that it has an.instance vector viewer which can give.you hints about spectral overlaps.between different fluorophores.already before running the experiment.how this actually works we will get back.to later in this session so looking at.the first panel plotting and gauging.strategies can vary from person to.person and there is really no right or.wrong way to go about it for me I like.to have a forward side scatter plot to.start with and we're looking for the NK.NK T cells.I start by gating the lymphocyte.population as this is where they both.the NK and a k NK t cells reside after.this plot do one or several Doppler.discrimination plots by plotting for.instance forward scatter area versus.forward scatter height this allows us to.get to gate out the cells that stick.together and which can make the analysis.more complicated and it has to be.topless for instance might trick the.user into thinking that cells Express.markers that they shouldn't when in.reality it was just two cells passing.the lasers sticking together as one.event when we come to the fluorescent.markers we can start with C d3 as this.is the differentiator between the N K.and n K T cells as the NKT cells are cd3.positives.while the N case.are not and I will get back to this.point on numerous occasion during this.presentation but it's always a great.advantage if you know the biology behind.the self that you are identifying in.order to know what you're looking for if.we start by looking at the NK panel we.can see that it is a bit of uncertainty.in the CD 56 CD 57 plots in this case.the CD 57 positive is lower in CD 56.expression than the CD 57 negative this.is unexpected as normally this should.not occur a reason for this strange.expression pattern could be due to our.suboptimal panel where we have a lot of.disturbances due to bad bad fluorescence.choices another reason can be.contamination of other cells like B.cells dendritic cells and other cells.which are still present in the cd3.negative population if we look at the.cd3 positive NK t panel we can see that.the cells we can see that we do not have.high number of CD 56 positive cells and.when trying to identify it subsets in.the cd8 verses CD 57 plot it's pretty.much a lost cause as there are no real.distinct populations meaning we should.try and improve our panel by looking.further at other panel design parameters.so far we have looked at the instrument.and fluorophore availability but nothing.else we basically just picked what we.could find and made a panel out of it so.next step is to also take the.fluorophore brightness into account.normally one ranks the fluorophore.brightness from 1 to 5 or somewhere from.very weak to very bright but there is no.formal way of doing it if we look at the.histogram plot on this slide we can see.lymphocytes from human blood which have.been stained with different fluorophores.what this tells us is that our decision.when it comes to which floor for us to.use will affect the outcome of our.analysis as it will give better or worse.separation of the population depending.on their brightness so in this case we.can clearly see the different floor for.separating the CD 56 positive population.differently flora for brightness depends.on how many photons of firfer emits when.being excited by a laser and also the.conversion rate of those photons when.they hit the detectors and are converted.into electrons to conclude the histogram.panel we can see that alexa fluor 700.which is a dim or weak fluorophore will.not manage to separate the CD 56.positive population very well fitzy.which is the moderately bright.fluorophore will manage to a good job.but Alexa 4 4 4 88 which is also.moderate 4 4 will do it even better PE.which is a very bright fluorophore on a.system with the yellow green laser will.give the best separation on the CD 56.population getting a good separation of.your population is key as it makes.analysis a whole lot easier and can give.you some leverage when it comes to.trade-off with other fluorophores that.you might want to include in the panel.if you have the opportunity to work with.several markers it will usually improve.the possibility to identify populations.as all of a sudden we're using more than.one dimension as you can see in the dot.plot panel we still use the same CD 56.staining but on top of that the cells.are also staying with CD 8 Pacific blue.and with this extra marker it is now.fairly easy to identify the CD 56.population what this tells us is that.for four choices we make are important.for the outcome of the panel and now as.we have looked into fluorophore.brightness.should follow up by looking into another.important parameter namely antigen.density antigen density is another.important factor as the number of.antigen or target molecules that a cell.carries will correlate directly to the.intensity of the positive population in.the example given we see three plots.with different antigens staying with the.same p floor for all of them will give.different intensities as the number of.receptors differ for each marker for.instance in the top plot staying with.cd45 are a sharing more than 200,000.surface receptors it will show a strong.intensity for the positive population.cd5 in the middle plots with around.90,000 antigens give you a decent to.intermediate intensity while CD 56 with.around 10,000 surface antigens will give.a very low expressing dim positive.population this will of course effect.your panel as well so knowing the.antigen density of your marker is.crucial for panel design this example of.differences in antigen density points.out the necessity to also look into.antigen density when designing a panel.how do we use fer for brightness and.antigen density to build a better panel.our rule of thumb is that you try to.pair bright bright fluorophores.with low expressing antigens and vice.versa of course if you have the choice.you can pair bright fluorophores with.high expressing antigens as well but try.to avoid appearing low expressing.antigens with weak for a force as this.will make it very difficult to resolve.the dim positive population from the.negative ones now that we have covered.four out of five fundamental parameters.we can look into how a panel possibly.can look now when we also take floor for.rightness antigen density into.consideration.to make it easier for ourselves we can.go back to the initial NK mg T cell mind.map the difference is that now we will.also color code the different markers.according to antigen density bright.orange would be highly expressed.antigens like C D 3 and cd8 milder.orange equals intermediate antigens such.as C D 57 and no color correlates to the.low expressing antigens and if you do.not like color coding why not do it in.another way perhaps with the use of.symbols there is no right or wrong the.only thing that matters is that you can.easily tell the degree of the antigen.density but how you choose to do it it's.up to you for me I like color coding so.in this example we'll stick to that what.I choose to do for this example is to.keep the antigen density to 3 levels low.medium and high expressing for flora for.brightness I do the same so for this.example there will be three degrees of.lore for brightness weak moderate and.bright Illustrated as different shades.of green now what we will do is start.pairing the bright flora first with the.low expressing antigens and vice versa.I call the process of pairing according.to fer for brightness and antigen.density to balance your panels which I.think sums up the process pretty well a.suggestion when it comes to balancing.your panel is to start with the low.expressing antigens as it is most.important that they get paired with the.bright floral force so if we started.with CD 16 which is a low expressing.antigen we can pair it with the bright.APC then we take CD 56 and so it is also.a low expressing antigen and pair it.with the bright PE but already now we.have covered low expressing antigens and.we can move on to the moderate or.intermediate antigens which in this case.is CD 57 s CD 57 ISM is a moderate.expressing antigen so we can pair it.with the moderately bright six.and now we only have the high expressing.antigens left to pair with the.fluorophores but they are not as crucial.as the low expressing antigens so we can.more or less pick whatever floor we like.so we pair CD 3 with pacific blue and we.pair CD 8 with PE alexa fluor 750 and.now our second panel is complete so now.that we have balanced our panels we.could just go ahead and run the.experiment and do the analysis but do.you remember that was something else.that potentially was corrupting our.first panels we had a lot of NK.unrelated cells in the cd3 negative.population like B cells dendritic cells.monocytes etcetera this can lead to an.increased unspecific binding as some of.them have FC receptors and it can also.increase the spread of the negative.population due to out the fluorescent.characteristics so now when we have a.chance why do we not add a dumping.channel to get rid of these also it is.always appropriate with the viability.marketing flow cytometry regardless of.application since dead cells tend to.express all kinds of unexpected.receptors release intracellular proteins.and leaked DNA which makes the dead.cells sticky and increases the risk of.on specific binding to their surface as.well since we have plenty of free unused.detectors we'll add one of those bytes.as well so we add the viability and a.dump channel into the equation for the.viability marker we will use the vivo.fix at 353 for 242 and when it comes to.the dump channel it is not always.established which level of antigen.density it has as normally used several.different markers for different cells in.general the lineage markers that are.used for dumping channels have quite.high antigen density levels and are easy.to detect but as we do not know for sure.let us just assume that at least one of.them could be low to moderate and we.should therefore be paired with a.moderate to bright fluorophore.so let's pick plx offshore 647 as this.is the only bright fluorophore we have.left and by that we now have a panel of.seven colors that is considered balanced.when looking at the new panels we have.achieved two things since the previous.one number one on all our low expressing.antigens we use antibodies conjugated to.bright fluorophores and number two we.have excluded cells that were not.relevant to our analysis by including a.dumping channel and the viability die.just like in the previous panel we start.with a forward side scatter plot and do.Doppler discrimination but this time we.also have the viability marker to.exclude dead cells this reveals that.whole blood in general does not have a.lot of dead cells however if the sample.would have been mistreated if the SE.itself came from another tissue or.cultures there is no guarantee that this.would still be the case so always make.it a habit to make room for a viability.dye the viability analysis is followed.by exclusion of cells that are not.interested in by a dumping channel and.on the same plot we also put cd3.expression to identify which cells that.belongs to the cd3 positive and the cd3.negative fractions now looking at the.cd3 negative and k panel we can see that.our CD 57 positive cells are also CD 56.positive which was expected from the.start we can also conclude that the CD.56 positive CD 57 negative cells in.general are positive for CD 16 which is.also expected all-in-all balancing your.panel will be good enough to identify.the NK subsets in this case.however looking at the city three.positive NKT panel there is a slight.increase in number of CD 56 positive.cells but it's still problematic to make.a good distinction between positive and.negative population even if we use a.bright fluorophore like P in this case.for the CD 56 marker to try and rescue.the experiment we can also make use of.an MMO controller a fluorescence minus.one control where basically all the.colors except the CD 56 PE is included.in a stained sample this control would.allow us to identify the spread in the.PA channel that is caused by all other.fluorophores and also be used as a tool.to identify the exact point of where the.borders between CD 56 positive and CD 56.negative lies however the lack of.distinction between the populations.makes it very difficult to set the gate.properly this is also shown in the CD 8.CD 57 plots where we see a surprisingly.large number of cd8 positive cells for.both CD 57 positive and CD 57 negative.populations in general this number of.cells and expression pattern is not.expected in the NKT panel and even if it.looks good at the first glance it is not.reflecting the truth of the NKT cells.subsets this points out another.important factor of panel design we come.back to it again knowing the biology.behind the ER panels in this case even.if we did do this as a mo control to.distinguish where to put the gate what.we see is most likely a contamination of.cd8 positive cytotoxic T cells as the.distinction between the CD 56 positive.and negative is quite weak in order to.say the least so in conclusion we have.improved the panel quite drastically and.if we were only interested in NK cells.we could probably stop our panel design.right here but we also want to identify.the subsets of the NKT cells which.brings us to the final parameter of.panel design for this webinar.the spectrum overlap spectral overlap is.what can be described as signals that.are detected where they are not supposed.to be it derives from photons from one.floor for that is being detected by the.wrong detector resulting in false.positive signals in the image you can.see that the emission spectra fitzy is.quite wide so that some of the photons.emitted by the fluorophore passes.through the 585 / 40 filter and ends up.in DP detector channels so where does.the spectrum overlap come from spectral.overlap occurs through mainly three.mechanisms the first is using floor.offers that are on neighboring detectors.it might not be a problem if the filters.are far apart but the tendency with more.and more colors coming into play is that.also the filters get closer and closer.to one another if you have two floors.with a fairly close emission wavelength.also expect to encounter certain degree.of spectral overlaps in this example.given we look at the lexer floor 6:47.which has an emission maximum around 660.nanometers and alexa fluor 700 with an.emission maximum around 720 both of.these are on the red laser line it is.clear that the alexa fluor 647 bleeds in.quite drastically into the alexa fluor.700 detector but also the alexa fluor.700 slightly bleeds into the alexa fluor.647 detector so when looking at the.plots in the lower panel it is actually.only staying with the alexa fluor 647.and not alexa fluor 700 but it still.seems like the population is positive.for both for a force and this is a.result of photons from alexa fluor 647.ending up in the alexa fluor 700.detector.to manage false positive signals you.need compensation which means.subtracting the false positive signals.from the wrong detector this is done.statistically and brings the alexa fluor.6:47 population down to a single pass.this state where the median value of.this alexa fluor 700 of both the.positive and a negative population.should be similar a good panel design.with only a few colors can eliminate the.need of compensation completely and if.you have a lot of colors it will still.reduce the compensation needed thus.maintaining good resolution of the flops.another factor that introduces spectral.overlap is if you use colors with.similar emission spectra most colors.have a tendency of more or less multiple.laser excitation which means that as can.be seen in the figure if you use Percy.piece i 5.5 with an emission of.approximately 680 nanometers on the blue.laser you will also see signals in the.yellow-green laser for the piece iphone.5 detector and even the red laser in its.APC detector the same situation occur.with the PSI 5.5 excited by the.yellow-green laser and with an emission.maximum at around 690 it bleeds into.both other lace lines potentially.reducing the resolution of Percy psi 4.5.and a PC also for a PC on the red laser.you can see a spectral overlap into the.yellow green laser not as severe as the.other two but potentially still a.problem if you're looking for something.that requires a crisp resolution to be.detected finally in this slide for the.PSI 5.5 ferrochrome you can also see a.third reason for spectral overlap the.tandem conjugates which gives us a.signal not only around the 690 nanometer.on the yellow green laser line but also.around the 585 nanometer area where we.normally find the P 4 4 and ours also.there is a slight signal on the similar.wavelength on the blue laser and the.next slide will explain this more in.detail at.tandem conjugate is a kind of lure that.bridges the excitation of the molecule.by using a base Flores foundation of the.mission for instance PE alexa fluor 647.uses the PE to excite the elixir for 647.where the alexa fluor 647 itself do not.have the capability to be excited by the.five six one laser the principle is as.follows if we start with PE it uses the.561 or yellow green laser to catch.photons using the same wavelength it.will take up the photon and try to hold.them but will later end up emitting them.but at that time they've lost some.energy and the photon that they.submitted now has a longer wavelength of.585 nanometers but the photon initially.started with the wavelength of 561.nanometers and then it ended up with the.585 nanometer wavelength so with the.tandem conjugate we coat the PE molecule.with alexa fluor 647 molecules like.mentioned alexa fluor 647 molecules.themselves do not have the capability to.get excited by the five six one laser.but when coated onto the P molecule they.will hijack the 585 nanometer photon.that is submitted by PE and excite.itself when Alexa fluor 647 takes up.this 585 Photon it will later emit.another photon at around 667 nanometers.instead of the PE s initial 585.nanometer this process of hijacking.energy or photons in this case is called.friend fluorescence resonance energy.transfer and is dependent on distance.between the molecules spectral overlap.is one of the downsides of using tandem.conjugates as some of the B photons.escapes and bleeds into the P detector.thus creating spectral overlap taking.with cross laser excitation and tendon.conjugated into account and looking at.the P Alexa fluor 647 we can see its.effect on all laser line and also on the.same laser line with the base of the.tandem conjugates in reality it means.that we have false positive signals on.all these laser lines that potentially.will reduce the resolution of our.populations by spread on the negative.population and it will also requires.compensation to subtract these false.positive signals if we look at the most.severe spillover it occurs in the.neighboring red laser and descibe v.channel on the blue laser the two bottom.plots on the slide where a line.illustrates the need of drastic.compensation which potentially can harm.the resolution of the population if you.want to learn more about compensation.and controls to use please see the.previous webinar from michael blundell.where he covers just these topics it can.be found on a WWI red antibodies comm.slash webinar has already established.the consequence of spectral overlap is a.reduction of resolution and the need for.compensation in this slide we see a plot.from an eight color panel which is.currently uncompensated for this.particular example as the markers were.looking for our CD 3 on T cells and CD.19 on B cells which are never Co.expressed on the same cell it means that.in reality it's not too big of a problem.to identify the separate populations.even when the data is uncompensated.it is fairly straightforward to identify.the t-cells positive for cd3 close to.the y-axis and the cd19 positive cells.looking like double positive due to.spectral overlap from one of the other.fluorophores bound to be so after.compensation you can see that the cd19.positive population has moved down on.the plot to do to subtraction of the.false positive signals from the cd3.detector but using the dotted line as.reference you can also see that the.spread of the cd19 positive population.is wider than the negative meaning a.loss of resolution the danger of this is.for instance if we're using a histogram.to identify the cd3 population what.happens in this case is that all the.cd19 positive cells that are above the.dotted line will be included in the cd3.positive population giving us a biased.cell number if we just tilt the plot to.make it the same as the y-axis on the.dot plot it would be even more obvious.what has happened since we only look at.one dimension in the histogram we have.no means of knowing what this is spread.from the B cell population that will end.up in the cd3 positive fraction in this.case the t cell population differs by.1.5 percent between histogram and the.dot plots but depending on how severe.the spread is this can be more or less.crucial for obtaining good reliable.results also if there actually were a.population that was double positive for.the two markers not even dot blots would.save us from making a mistake it is also.important to know that both the spread.and compensation is a function of a.slope this means that when we when we.compensate for something that is really.bright for instance and for instance.using compensation beats everything with.the lower intensity will be compensated.as well the same goes for spread to.higher the intensity of the population.to bigger the spread.if we just illustrate it with the.triangles you can see how the spread.would increase as the intensity of the.positive population is increased grid.red circle illustrates our current see.the 19 positive population that means.that if we would have had higher.intensity of the population the spread.would be more severe if we like in the.case had the cd3 and see the 19 that is.never expressed on the same cell it.would not be a problem but if it was.another cell type that expressed both.fluorophores this is what would happen.the spread of the negative would bleed.into the double positive population much.more severe than with the lower.intensity just making it almost.impossible to make a reliable analysis.however this is more advanced panel.design and we shall not die further into.this in today's presentation if we take.spectral overlap into consideration when.designing our panel we can actually.conclude that the panel - already looks.quite good there is not really any.issues with detecting the NK cells at.this point but finding the subsets of.the N key T cells was a bit problematic.so where can this problem come from if.we look out of a spectral overlap.perspective the most likely reason for.the difficulty to get a good 356.resolution is due to spillover by PL x.f4 750 on the cd8 cells into the CD 56.PE channel so we need to find a panel.where the fluorophores conjugate.conjugated to the cd8 and CD 56.antibodies do not cause this severe.spectral overlap which means that to.make the panel as good as possible.actually only a small adjustment is.needed.in general P is a nice and right.fluorophore so I would not like to.replace that one since it has good.potential to lift out the dim positive C.D 3 C 256 positive cells from the.negative population so let us instead.focus on the choice of the fluorophore.of the cd8 antibody as it is a.high-density antigen thus giving us a.little more options on which floor force.to change without losing too much.resolution at this point a good later to.pick the fluorophore from would be the.violet laser as we currently do not have.too many flowers there and it's a quite.small risk of spectral overlap the only.color we can choose a specific blue.which is currently occupied by the cd3.antibody since we now have to find.another floor for cd3 alexa fluor 700.would be a good choice yes it bleeds.into the APC channel but on a PC we have.cd16 our Markel which is not currently.of interest for the NKT cells and even.if it potentially could reduce the.resolution of CD 3 we would still have.the cd8 expression to further.distinguish these cells if necessary so.this leads us to a panel that out of the.situation given would take all parts of.panel design into consideration now when.looking at the list of first it seems.like we have them pretty well.distributed over the lasers and with the.decent separation of emission spectra of.the fluorophores.this is one of the key points when.designing at the panel as it potentially.will reduce the spectral overlap and.also reducing the spread looking at the.spectrum viewer we can see that some.color still bleeds into other detectors.but let me explain why this is not.really a problem in this case one.possible problem could be specific blue.bleeding bleeding into the vivo fix.detector and the UV laser like mentioned.previously spectral overlap will only be.an issue if the two fluorophores are.co-expressed on the same cell in this.case one of the colors is a very.viability marker and all cells.expressing this color is going to be.excluded from.the analysis as they are dead this means.that when we have a situation when these.two two colors are in fact co-expressed.it will anyway be discarded from the.analysis the same goes for our dumping.channel conjugated to P Alexa for 647.which leads into a PC it is just as for.the viability channel that when we have.Co expression of a PC and PE Alexa fluor.647 those events will be excluded from.the analysis as they are from a cell.from a non relevant lineage and will not.be taken into consideration so let's see.how the final panel turned out so just.like previously we start with the.forward side scatter plot and do the.Doppler discrimination before getting to.the viability staining there are still.not too many dead cells in this sample.as it is from freshly drawn blood and.with the dumping channel we get rid of.most cells that are relevant for NK NK T.cell analysis on the same plot that's.the dumping channel we also have the cd3.staining which is used as the.differentiator of the CD three- NK cells.and the City 3 positive and key T cells.we can see that even with with the APC.on CD 16 potentially bleeding into the.CD 3 channel it is still easy to.separate the positives from the.negatives when looking at the CD 3.negative NK panel we can see that the.cells still look good and it's easy to.identify the populations that we.initially set out for also now with our.new panel when we look at the NKT cells.we can see that the resolution of the CD.656 has improved which makes it easier.for us to gate on the CD 56 positive.cells this results in a much better and.KT subset plot of the cd8 verses CD 57.where the resolution is good enough to.identify the slight intensity.differences for the six subsets we.mentioned earlier.now if we look at the improvements that.the panel design has made for our n K.and K T panel we can see that just.picking random floor first like we did.in panel 1 is most likely only causing.confusion and in the end no real.conclusion can be made when taking fir.for brightness and antigen density into.consideration as seen in panel to some.but not all of the populations can be.identified especially the resolution of.C D 56 is the limiting factor of the N.key T subsets in this panel whereas in.panel 3 when we're using all the rules.of panel panel design building we can.identify all the NK cells and also all.the entities subsets that we initially.set out for.so in summary of panel design always.start with looking at what instrument.you have access to and which filters.detectors that the configurations offer.then continue to check which antibody.fluorophore choices you have balancing.your panel by combining bright floors.with dimension and vice versa will get.you a really long way take spectral.overlap into consideration and try to.choose fluorophores across lasers and.with different emission wavelengths read.up a bit on the panel you're trying to.design it is easy to make mistakes as.flow cytometry will always produce data.for you but it's your job to make sure.that the data is valid some other tips.and tricks to help you achieve good.quality data of your flow cytometry.experiment number one is to always.titrate your antibodies and do it for.each individual type of sample and.according to the starting number of.cells by titrating not only will you.reduce the spread of the negative.population and to limit the risk of.unspecific binding of the unbound.antibodies but it will also reduce the.cost of antibodies as a vial will last.longer and use controls.there are numerous controls out there.and you should at least at some stage of.your experiment have tried the ISO type.controls to identify possible and.specific binding of your antibodies you.should also use MMO controls that can be.used not only to identify the border.between the positive and negative.populations in your panel but you can.also use them to trace which fluorophore.that causes a spread for another.fluorophore on top of that or biological.controls negative controls mob controls.all of them are important and if you're.not sure please check out the resources.on the by red antibodies web page for.more detailed information and finally.consistent consistency it is more.important than a completely perfect.panel you can get a long way even with.the decent panels so even if you learn.today that your current panel is not.perfect this is not the reason to start.changing your panel now you should.finish what you started so that all of.your data is comparable and then for the.next experiment you can start.redesigning your panel to make the.analysis easier on top of that never.like just add another color to on the go.as you learn today this will also alter.the balance of your panels and it might.not be comparable to your previous.experiments anymore so avoid doing it.until the time it is time to start with.a new experiment or project for more.information on flow cytometry including.building multicolor panels controls.protocols fluorophore charts gating.strategies and an updated flow guide.covering the basic of flow cytometry go.to WWII tenta bodies comm /resources.there you will also find resources for.other antibody-based applications such.as Western blot immunize the chemistry.Eliza and immunofluorescence in addition.to this flow cytometry panel design.webinar in our resources section we have.three other flow cytometry webinars.including one on controls essential for.all flow experiments we also have.application based webinars on topics.such as mastering immunohistochemistry.experiments and immuno proceed.and various research area webinars.covering areas such as apoptosis all.available to view on demand the last.slide today is to inform you of all the.antibodies reagents controls and.instruments we have here at bio-rad to.help you with your flow cytometry.experiments antibodies both single-unit.and cocktails with a range of.fluorophores viability dyes.proliferation dyes and easy to use.apoptosis kits are available go to.bio-rad antibodies comb slash flow for.more details and this brings me to the.end of today's webinar and I would like.to thank you for listening.Thank You Sebastian for that interesting.and informative presentation and now I'd.like to open the Q&A session for this.Q&A session Sebastian will be joined by.Sharon Sanderson Sharon is a flow.cytometry research associate here at.biro.Sharon obtained her degree in molecular.biology from the University of.Manchester before moving to the.Institute of cancer research obtaining a.PhD in cell biology from the University.of London with her thesis focusing on.angiogenesis she then moved to the.University of Oxford where she worked.for 13 years initially continuing with.research in the cancer field before.moving into immunology research her most.recent position was a postdoc in an auto.Phase II laboratory working on multiple.projects involving conventional and.imaging flow cytometry Sharon moved.earlier this year to join the bio-rad.team as a flow cytometry research.associate working on the new z d5 cell.analyzer and now over to you Sebastian.and Sharon alright so we did get a few.questions during this session so what I.will do is that I will start answer a.few of them then I will hand over to my.colleague Sharon so first one do I.really need to follow all these panel.building rules for a three to four color.panel or can I use this straight out of.the fridge panel design it kind of.really depends on your panel but in.general what you can say is that the.more colors you have the more crucial it.is that you use these kind of design.tools make your panel you might get.lucky if you use three to four covers.because you have a lot to choose from.but in general if all of them are low.expressing antigens make sure that you.have at least right Laura compare them.with otherwise even three to four colors.is going to be really difficult to.analyze next question if I have.be separated two cell types by mutually.exclusive markers on T and B cells can I.split one fluorophore over to two other.mutually exclusive markers in those.lineages further analyze them so the.question is for instance if I have one.market for a T cells and one market for.a B so could i for instance use PE.marker for both of them.as I can use these initial markers to.separate them and yes you can do this.the only thing that is important is that.you use appropriate controls and as you.do compensation on the brightest of the.two population you also have to make.sure that you don't have on specific.binding between the two populations by.doing a staining test with with each one.of the two antibodies separately kind of.like an MMO but you don't really exclude.any of the torah' force just the first.one and then the other one and just.check that you don't have any other.binding and you cannot however use.different fluorophores so for instance.if one of the markers is spawned let's.say APC and the other one is on Alexa.for 647 both of them would share the.same detector but the compensation.values would be different so it would.kind of skew your panel but it's not.recommended it has to be the same.fluorophore if you want to use method.and not a question and I'm actually.going to combine this with yet another.question so it's two questions in one.and so once I have done my compensation.and set up my matrix do I need to repeat.the single tubes for every experiment.meaning do I have to compensate every.time and the other question was how.important is it to include unstained.cells also in the knowledge in the.analysis and this unstained question is.about I would say it's a bit about.interpretation because for analysis.itself the unstained tube is not very.crucial but it could give you a lot of.information about out the fluorescence.and how to set your voltage and so on.before doing compensation so it should.be included during the setup process for.the analysis part and you can do without.it.and when it comes to reusing.compensation over and over again and I.first want to say that it's it's never.wrong we would always do a fresh.compensation because whenever you do a.new compensation always takes the given.situation so the temperature the.fluorochromes are using everything in to.account for that very specific situation.we always get the best compensation out.of it but if you find it very annoying.we've always worried compensation what.you can do is before starting your.experiment you go back to the previous.experiment where you where you record it.and calculate the to compensation and.you checked what what the voltage values.if my QC tells me that they and then for.the current day go into the QC and you.check what voltage did I use today.if those voltage is more or less.correlate with only a few volts.difference most likely yes you can reuse.your compensation however if it has been.fluctuating your instrument you might.really need to do a recompensation it.really depends on on on your instrument.how it's performing and also keep in.mind that if you use tandem conjugates.those can degrade when exposed to light.and overtime so it might be a good idea.to double check your talent conjugates.if you want to reuse a compensation for.a panel this is including and impunity.and but there is really no right or.wrong thing to do just make sure that.you check before starting if if you can.use your compensation or not but in.general if you use standard for a force.like PE APC HB those fluorophores they.don't degrade too quickly so if your.panel only using those goes for a force.most likely you can reduce operation.done and so I'm.I'm going to hand over to my colleague.Sharon so she can answer a few of the of.the questions as well hey Thank You.Sebastian so we've had a question come.through asking if you can combine.extracellular and intracellular markers.in a panel so yes there's absolutely no.problem with doing this but you do have.to stain first for the surface markers.and that's because the perm.immobilization required for.intracellular staining can affect.surface staining you also need to think.about the sample fixation step so.whether to fix before surface staining.or after and that's because some.antibodies did you know don't stain very.well and on em once cells are fixed.rather than a month X finally you should.also consider if the flora for use of.your surface marker is compatible with.subsequent fixing and / Malaysian.there's some optimation up optimization.of your staining panel may be required.for example tandems and large floors.could have to be fixation sensitive do.you'd like to avoid these so the next.question we've had through is how can I.check the performance of my tandems.and monitor them for breakdown in fret.the easiest way to do this is to check.your Tam tandems using your single stain.control they're using the single single.stain control from compensation you can.check the amount of fluorescent from.both the donor floor and the acceptor.floor for if we take the Alexa six or.seven as an example.is there more fluorescence in the PE.channels at 578 nanometer from the five.six one laser compared to six six five.nanometer from the six forty laser then.the tandem may have degraded so we've.had quite a few questions actually.asking about antigen densities so you.want to know where can you find antigen.density information but the best place.to look for this if is if you do a.literature search as is actually there's.quite a lot of published data already on.this.so I'm just going to take one last.question so we've had a question asking.which is the best brightness of flora.for for an antigen that changes its.expression levels and in an experiment.so something like an activation marker.so in this case I would choose a bright.flora for this will enable you to get.the best separation of signal from your.lower expresses compared to the negative.cells also it's probably best to choose.a floral with little spillover or cross.laser excitation to ensure you can see.two positives positive and negative also.you should don't forget yet to run your.FMO control so you can actually set your.gate so this now brings us to the end of.today's webinar and hope the nail you're.more confident at designing multicolor.panels if you want any more information.please look at the bio red flow guide.which has tips about panel design and.other flow topics and many thanks for.listening today.

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How can I fill out Google's intern host matching form to optimize my chances of receiving a match?

I was selected for a summer internship 2016. I tried to be very open while filling the preference form: I choose many products as my favorite products and I said I'm open about the team I want to join. I even was very open in the location and start date to get host matching interviews (I negotiated the start date in the interview until both me and my host were happy.) You could ask your recruiter to review your form (there are very cool and could help you a lot since they have a bigger experience). Do a search on the potential team. Before the interviews, try to find smart question that you are Continue Reading

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