I'm at home, so somewhat limited in my ability to open some of your attachments I'll give them a whirl in the morning, however the first pdf is interesting so I'll address item 1. What are you using to plot the FACS Calibur data? What are you using to compensate the FACS Calibur data? What does it look like if you compensate it using the calibur? What does it look like if you plot it in CellQuest? If you're using software to compensate 10 bit linearly-acquired data, then using software log transforms and /or software compensation rather than using the built-in hardware (as imperfect as it might be), I'd question your judgement :- there's a thick seam of artefacts there, and it's one that I'd mine first before worrying about machine "linearity" or dye metachromicity. I'd suggest you get try compensating using the hardware, see if the data looks weird still, if so ring your local product specialist and seek his or her advice. If it looks fine compensated by the machine and acquired in whatever flavour of cellquest you have, then get in touch with your 3rd party software vendor and seek advice. Cheers Ray ----- Original Message ----- From: "Uriel TK" <utk1@013.net> To: cyto-inbox Sent: Monday, March 26, 2007 8:27 PM Subject: 2 machines, 2 compensation troubles. Friends: As usual, when confronted with trouble I turn to you for help. this concerns two machines with similar problems. 1) The FACScalibur in our lab. I performed a titration experiment for Sytox Green and when checking the compensation needed for it to work with other stains, saw the results shown in the pdf "SytoxG linearity". I have attached one of the files (ApoPMN.NucAcTit.1mar07.021) if you want to check it out. As you can see, instead of being a nice distribution on the X axis, it looks like a waving serpent! On close inspection, the waviness can already be seen in the uncompensated FL1 vs. FL2 plot. After long thought, I have arrived to the conclusion this is due to faulty log-amp behavior causing deviations from linearity. Certainly, this has important consequences for our results on that machine! In order to confirm my findings I exported the FCS data to excel, sorted the data according to the FL1 intensity and did a FL1/FL2 plot. After accounting for statistical noise, the mean ratio should stay constant - after all, that is the rationale of compensation! As you can see from the "SytoxGreen.xls" plots, that is hardly the case. I turn to you to ask for alternative explanations and possible solutions. Is this kind of problem repairable? how easily? We have a service contract with BD for that machine, so would I be right to assume they should fix it? What kind of tests would you suggest to confirm and/or refine my findings? PS: SytoxGreen is NOT metachromatic AFAIK. 2) We just got a LSR II at our core facility, and needless to say I've eagerly started working on it. To my surprise, I have found erratic results when using the PerCP-Cy5.5 channel, aka FL3, off the 488 laser. If you care to open the "temporal2" pdf and look at the bottom plot, it is a compensated plot of DR-FITC. Once compensated, no matter where I set my gates, the Y value is very close to an exact match for the control, and they are all similar to each other, as expected since I'm correcting for the spillover = compensating. Now, if you look at the top plot, a different situation is happening: This is a PI stain, and I've compensated the gate K1 with the unstained cells. The other gates are not compensated! moreover, the plot is all skewed: although the median for the gate K1 is of the same value as the unstained, the population is clearly not symmetrical around it. It seems as if there is a significant deviation from linearity somewhere above the 10^4, causing a distortion of the ratio of the two channels and precluding proper compensation. I did the same plot as I used for the previous case in "comp analysis UDC53.xls" . As you can see the FITC compensation's center stays in the same place. The PI one shows a clear drift. I have attached the two fcs files also if you want to play with them. Furthermore, in a following experiment I titrated lower concentrations of PI hoping to avoid the linearity "kink" and thus be able to use PI. As you can see in "temporal1.pdf" I did attain better looking distributions but the compensations still do not match at all heights; although the differences are relatively small, they shouldn't be there to that extent. I do grant that I had small samples for this second experiment and there is the possibility for having too little cells for good math. In any case, you can see that the same compensation setting for the same dye does not properly compensate the different samples! As before, the same questions apply: what is going on? PI is certainly not metachromatic! Is this problem also related to linearity errors? What kind of tests would you suggest to confirm and/or refine my findings?All input is dearly appreciated. Finally, this experiment was an expensive and extensive one, and I'd hate to loose my data due to PI's uncompensability. I've been thinking, and maybe I can use the "PI only" sample to plot the deviations from linearity and use them as a template to "pre-compensate" the other files so that I correct the data, and thus use the corrected files for my analyses. I use FCS Express which has parameter math capabilities (I can apply the parameter math and create a new parameter thus not destroying original data). How would you suggest I proceed for the corrections? Another option would be the "brute force" approach: create ~6 gates at different levels of PI intensity, calculate the compensation needed on each one, apply the differential compensations at the different stages and recreate a plot consisting of the 6 gates joined together. Although that would not perfectly match the deviations it would minimize them to a workable situation. Thanks beforehand for your much appreciated help, Uriel. Uriel Trahtemberg, M.Sc. MD/PhD student The Laboratory for Cellular and Molecular Immunology The Hebrew University - Hadassah Medical Organization Jerusalem - ISRAEL "When you were born, you cried and the world rejoiced. Live your life in such a manner that when you die the world cries and you rejoice" Old indian saying -------------------------------------------------------------------------------- > This attachment - '24 hrs_35 - 25LPS - DR FITC.fcs' - 241.66 KBytes - can > be viewed at > http://www.cyto.purdue.edu/MD-parts/97375482258482ca6498e05f90d5205c5d3107b6.fcs > > -------------------------------------------------------------------------------- > This attachment - '24 hrs_14 - Ctrl - PI only.fcs' - 241.66 KBytes - can > be viewed at > http://www.cyto.purdue.edu/MD-parts/6a25c597ab762e775a4d3dbcd8e889c782ca0200.fcs > > -------------------------------------------------------------------------------- > This attachment - 'comp analysis UDC53.xls' - 964.10 KBytes - can be > viewed at > http://www.cyto.purdue.edu/MD-parts/9fb3d2f9ceb93313faa516bb57477018e7db7ed5.xls > > -------------------------------------------------------------------------------- > This attachment - 'SytoxG linearity.pdf' - 28.80 KBytes - can be viewed > at > http://www.cyto.purdue.edu/MD-parts/d3a970b7c8c072408253843372084ffaff35a4d6.pdf > > -------------------------------------------------------------------------------- > This attachment - 'SytoxGreen.xls' - 132.61 KBytes - can be viewed at > http://www.cyto.purdue.edu/MD-parts/05a5793cb6ee2f44b6ae44073c291c0da1876457.xls > > -------------------------------------------------------------------------------- > This attachment - 'ApoPMN.NucAcTit.1mar07.021' - 155.13 KBytes - can be > viewed at > http://www.cyto.purdue.edu/MD-parts/a5349f1884bf91c59aee53feb3c830411d1dabb5.021 > > -------------------------------------------------------------------------------- > This attachment - 'temporal1.pdf' - 38.31 KBytes - can be viewed at > http://www.cyto.purdue.edu/MD-parts/928a561f6e6b75c3d20a2ddc9a18c3fa403ba0ab.pdf > > -------------------------------------------------------------------------------- > This attachment - 'temporal2.pdf' - 17.72 KBytes - can be viewed at > http://www.cyto.purdue.edu/MD-parts/eeb2be1801bb1558098bc32a2061b7ab829cc9f8.pdf > >Received on Wed Mar 28 14:38:00 2007
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