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1、CELL CYCLE ANALYSISCELL CYCLE ANALYSISAnalysis of a cell populations state of replication can be achieved by fluorescently labeling the cell nucleithen analyzing the fluorescence properties of each cell in the population by flow cytometry. Quiescent andG1 cells will have one copy of DNA and will the
2、refore have 1X fluorescence intensity. Cells in G2/M phaseof the cell cycle will have two copies of DNA and accordingly will have 2X intensity. Since the cells in Sphase are synthesizing DNA they will have fluorescence values between the 1X and 2X populations.1X2XSG0/1 = 1XS PhaseG2/M = 2XDYE FLUORE
3、SCENCEThe resulting histrogram consists of three populations: two Gaussian curves (1X and 2X peaks) and the S-phase population. Adjacent populations overlap each other. Because of this, a modeling program is requiredto de-convolute the populations and assign percentage values to each population. Exp
4、ert and subjectivereview of the modeling softwares cell cycle phase percentage assignment is the final stage of cell cycleanalysis prior to reporting the results.G0G1: 33.94 % Mean: 49.19G0G1G2M CV: 2.97 %S-PhaseG2M: 15.28 % Mean: 96.41 G2/G1: 1.96S-Phase: 50.78 % Mean: 69.20R1R10030609012030Channel
5、s (FL2-A)6090120FL2-WCopyright 2004 Nathan RegimbalCopyright 2004 Nathan RegimbalPULSE PROCESSINGPULSE PROCESSING=AGGREGATES: A STICKY SITUATIONAGGREGATES: A STICKY SITUATIONFLOW CYTOMETRY AND CELL CYCLE DATA: DUE DILIGENCEFLOW CYTOMETRY AND CELL CYCLE DATA: DUE DILIGENCEAs cells transit through the
6、 laser beam, their fluorescence signals ultimately generate voltage pulses by thefluorescence detector (photomultiplier tube (PMT).W322LASEREXIT BEAMMID BEAM =1MAX SIGNALCELLSIGNALENTERSAREA13BEAMTIMEThe resulting pulse has three measurable features: height, width, and area.Height = maximum fluoresc
7、ence intensity. Width = transit time. Area = total fluorescence of particle.Special considerations must be taken when optimizing a flow cytometer for cell cycle analysis. Because theDNA histogram is the final product of the flow cytometer that is subjected to curve de-convolution analysis,the integr
8、ity of the data must be high and the fidelity of individual cells fluorescence must be maintained.This is mentioned because of the simple nature of cell cycle analysis: looking at events with distinctfluorescence levels. As mentioned before, cells in G0/1 have 1X fluorescence and G2/M cells have 2Xf
9、luorescence. What happens if two G0/1 cells pass through the beam (whether stuck together or not) at thesame time?As we all know, cells can stick to each other. Also, despite the hydrodynamic focusing that occurs at theflow cytometers laser intercept, multiple cells can pass through the laser at the
10、 same time even if theyre notstuck together.What results is the recording of one type of event as another type entirely. Because you are examining thenuclear fluorescence of each event, two cells in G0/1 that are stuck together will have as much nuclearfluorescence as one G2/M cell. This will result
11、 in analyzed G2/M percentages that are erroneously high.One G2 CellTwo G1 CellsAggregates and simultaneously read events are unavoidable during the acquisition part of cell cycle analysis.The way to maintain the fidelity of the data is to exclude these non-single cells from later analysis. This isac
12、complished by using the cytometers signal pulse processing and taking care to optimize the instrumentproperly.HCopyright 2004 Nathan RegimbalVIEWING PULSE PROCESSING PARAMETERSUsually, only the height (maximum fluorescence emission) signal is recorded (so THATS what the H inFL2-H means HEIGHT!) In c
13、ell cycle analyisis, however, all three parameters (H, W and A) must beaccounted for.Pulse width is indicative of particle transit time. Just as a tractor trailer takes longer to cross an intersectionthan does a compact car, single cells will have smaller pulse widths compared to cells that have agg
14、regated.Below is a histogram of the FL2 peak emission values (FL2-H). Alone, it can act as the subject of a de-convolution program. However, since FL2-H alone gives no information about total fluorescence orsingularity, it cannot be used for accurate cell cycle analysis.091900 SKW CC ARR.020CLOSE.Hi
15、stograms generated withCellQuestTM Pro from BectonDickinson02004006008001000FL2-HThis is a histogram of FL2-Area (total cell fluorescence). This graph looks VERY similar to the FL2-Hhistogram. Although it is showing us the correct parameter for DNA analysis, it still does not allow foraggregate disc
16、rimination.091900 SKW CC ARR.020BETTER.02004006008001000FL2-ACopyright 2004 Nathan RegimbalFL2-ACELL#THE RIGHT WAY TO EXCLUDE AGGREGATES: FL2-W vs. ATHE RIGHT WAY TO EXCLUDE AGGREGATES: FL2-W vs. AA REVIEW OF CYTO-ANATOMY: PMT VOLTAGE AND AMPLIFIERA REVIEW OF CYTO-ANATOMY: PMT VOLTAGE AND AMPLIFIERT
17、he left plot (below) shows pulse width versus area, and this is the plot used to distinguish between singlecells and aggregates. Single cells (G0/1 or G2/M) will have similar pulse width (transit time) values.Aggregates will have larger width values and can be easily seen on the plot to the right of
18、 the single cellregion.Single cells have been gated (left plot) and an FL2-Area histogram has been drawn and formatted to showonly the events inside of the single cell region.091900 SKW CC ARR.020091900 SKW CC ARR.020BEST!Note: If the single-cells arecontinuous with either thedebris or aggregates, y
19、oushould include them in theDNA analysis model. Theprogram is able to calculatehow the debris andaggregates overlap the singlecells and it will probably bemore accurate than you tryingto gate them out manually.SINGLE CELLS0200400600800100002004006008001000FL2-WFL2-AThese are the plots you will use i
20、n the analysis program. But before you get to that point, you must firstoptimize the flow cytometer to generate data that looks like this. How is this done?When a cell passes through the laser, a pulse is generated (see above). Assuming proper thresholdadjustment, the characteristics of this raw pul
21、se will be determined by one thing: the cell and the PMTvoltage.The PMT is like a mousetrap for photons. When the photons from a fluorescing cell strike the PMT, it beginssnapping. When a bright cell fluoresces, the PMT receives a big signal and therefore produces acorrespondingly large voltage puls
22、e. Because the PMT operates by way of its applied voltage, the gain (howhard it snaps at a cell) can be increased or decreased by increasing or decreasing the PMT voltage.By increasing the PMT voltage,you can change a pulses height,Low VHigher Vwidth and area values.PMT pulsePMT pulseWHAT AN AMP GAI
23、N DOESWHAT AN AMP GAIN DOESSometimes you need to move things around delicately on a parameter. You do it all the time with ForwardScatter. Ever notice that the FSC VoltageVoltage is adjustable only in large increments of 10 (E 01, E00, E01, etc)?In order to carefully move a population on Forward sca
24、tter, you need to take that rough voltage pulse fromthe FSC detector and change it slightly. This is done with the FSC Amp gain.Note that the forward scatter parameter is labeled FSC-H. The H stands for Height. The forward scatterparameter is showing only the height of the voltage pulse. What the am
25、p gain does is this: it takes the rawvoltage pulse from the detector and pre-amp (more machine guts) and it carefully amplifies that signal evenfurther. In practicality, the amp gain only affects a pulses Height value.No Amp Gain: Raw pulse from PMTAmplified 2x original (amp readout 2.0)Increase the
26、 amp gain to 2.0Doing so will double the2H outputheight of the raw voltagepulseHWHITE NOISEHThe amp gain on a flow cytometer ranges from 1.00 (no alteration of raw voltage pulse) to 9.99 (nearly 10times the amplification). On FSC, if you need to amplify more than 9.99, you adjust the rough voltage s
27、ettingto the next step (a ten-fold increase) and bring your FSC Amp gain setting back down to 1.00 and thenadjust your amp gain as necessary.An illustration of amplifier function involves electronic music playback. A CD player reads the media andhas line-level (low voltage) outputs in the back. Why
28、cant you simply connect speakers directly to a CDplayer? Although the signal is present in high fidelity, the amplitude of the signal is very small. So small, infact, that a speakers magnet wont be energized with enough electricity to vibrate and produce sound. Whatyou need to do is plug the CD play
29、er into an audio amplifier. The amplifier takes the original signal (read,PMT voltage pulse) and carefully increases pulses amplitude at every point of the signals waveform (read,amp gain). The resulting signal, having much higher amplitude, will now have enough “oomph” to power aloudspeaker. In flo
30、w cytometry, we dont need to add so much amp gain to our PMT voltage pulse that wecan power a loudspeaker, but we do add enough amp gain to inch it up on a parameter to make it look good.Speaker/Its kind of like thisPARAMETERAmplifier/AMP GAINCD Player/PMTSignalSIGNALCopyright 2004 Nathan RegimbalCo
31、pyright 2004 Nathan RegimbalSO WHAT DO I ADJUST TO OPTIMIZE FOR CELL CYCLE ANALYSIS?SO WHAT DO I ADJUST TO OPTIMIZE FOR CELL CYCLE ANALYSIS?Step by step optimization using CellQuest be sure to ask for expert assistance:Step by step optimization using CellQuest be sure to ask for expert assistance:Dr
32、aw the following plots: Two parameter: FSC/SSC, FLW/FL2A; Histograms: FL2-H, FL2-ASet your threshold on FL2 at 20. In the art of flow cytometric cell cycle analysis, it is standard to setthe threshold on the DNA fluorescence parameter at a value of 10% of the location of the G0/1 peak.The G0/1 peak
33、will therefore be placed at a value of 200 on the FL2-H parameter (see below).Select FL2 as the DDM parameter (lower right portion of the Detectors/Amp Gain window in CQ).Put FL2 in LIN amplification. Linear amplification is necessary to resolve the 1x and 2x fluorescencepeaks of the G0/1 and G2/M c
34、ells (respectively) on the FL2 parameter. If the amplification werelogarithmic, the peaks would be smashed together.Place the Amp gains for FL2, FL2-A and FL2-W at 1.00.Place a sample on the machine and run in LOW speed (low speed ensures lowest possible CVs andminimizes coincidence events)Because t
35、he threshold is set on FL2, no events will be seen unless the FL2 PMT has enough voltage togenerate pulses above threshold. Increase the FL2 voltage if no/few events are registering in themachine.Once the events are above threshold, adjust FSC and SSC as usual.The G0/1 peak will be discernable from
36、the G2/M peak. With a diploid control, you will see twodistinct peaks on the FL2-H histogram.Adjust the FL2 voltage so that the G0/1 peak has a value of about 100 on FL2-H.There should be a roughly similar profile at this point on the FL2-A histogram.Increase the FL2 amp gain (not voltage) to move t
37、he peaks up on the FL2-H histogram. Since theamp gain only adds height to a raw voltage pulse, the population will only move on FL2-H. The otherFL2 derived parameters (W and A) will be unaffected.Increase the FL2-A amp gain until the G0/1 peak has a value of 200 on FL2-AIncrease the FL2-W amp gain u
38、ntil the single cell population has a value between 200 and 600 onFL2-W.Adjust the FL2 voltage, amp gain and the FL2-W and A amp gains until the data appears similar tothe data below:Copyright 2004 Nathan RegimbalCELL#SSCCELL#FL2-AWHAT GOOD DATA LOOKS LIKE091900 SKW CC ARR.020091900 SKW CC ARR.02002
39、00400600800100002004006008001000FSC-HFL2-W091900 SKW CC ARR.020091900 SKW CC ARR.0200200400600800100002004006008001000FL2-HFL2-AGenerating data like this involves careful adjustments and readjustments of PMT voltage and Amp gainsettings.0200400FL2-W600Copyright 2004 Nathan RegimbalACQUISITION DETAIL
40、SACQUISITION DETAILS8001000Accurate de-convolution of the DNA histogram requires that at least 10,000 events are available for analysis.Because the single cells are the subject of analysis, its important that, during acquisition setup, a collectiongate is used to ensure that enough single cells will
41、 be stored. The best plot for aggregate/debrisdiscrimination is the FL2-W vs. FL2-A plot. Use this plot to draw a gate around your single cells. Thefollowing plot shows the single cell gate as well as the other populations on the plot.091900 SKW CC ARR.020Set Acquisition and Storage so that 10,000 e
42、ventsSet Acquisition and Storage so that 10,000 eventsDEBRIS/DIPLOID CONTINUUMare counted in the single cell gate. Be sure toare counted in the single cell gate. Be sure toaccept and store all events.accept and store all events.AGGREGATES091900 SKW CC ARR.020SINGLE CELLSDEBRIS02004006008001000020040
43、06008001000FL2-AFL2-WWhen analyzing the data in the cell cycle analysis program, use the single cell gate to exclude debris andaggregates - so long as they are not continuous with the single cell population. In the above plots, theaggregates and debris can be easily resolved. In the following plots,
44、 however, the aggregates, debris andsingle cells blend together. In this case, software debris/aggregate modeling is required to accurately de-convolute these populations in addition to the G0/1, S and G2/M populations. If high levels of debris andaggregates are present in your sample, accurate data analysis might not be possible.HCT 24h 350nM.003HCT 24h 350nM.003This could betough0200400FL2-A6008001000
