First, I would like to show my admiration and appreciation to this tool of flow cytometry mailing list, which make me feel that the whole world is one big country, and to all the people who make it great place to live in, thanks for the help in summary; * Everyone agrees that using bigger nozzle is essential in order to be able to look at all cells and not to squash them, this apply on both analysis and sorting [ I did used 150 ul, with Lin FSC and Log SSC, it was much better] * Trigger on FSC, allows me to see all cell sizes. * Display FL1 vs FL2 plot. Auto fluorescence normally runs on the diagonal whilst GFP SP cells will sit underneath and on this once compensation is applied. Lucy Brown from California and Dr Gill from Auckland both showed me how to do that, special thanks. * compensation using the reference gave to me by Rachel from Massachusetts Medical School ; Alberti S, Parks DR, Herzenberg LA.A single laser method for subtraction of cell auto fluorescence in flowcytometry. Cytometry. 1987 Mar;8(2):114-9. my next step * Using the beads recommended by Nathan at (www.QuantumPlex.com) is , B-D sells "FACS GFP Calibration Beads" Cat # 8375-. to set up a standard curve that relates fluorescence intensity to MESF (Molecules of Equivalent Soluble Fluorochrome) of EGFP. (a cell with an MESF value of 5,000 is as bright as 5,000 EGFP molecules) If I generate this curve, and calculate the MESF value of the "negative" cells, then it will help me to quantitatively the difference in expression * also Quenching auto fluorescence by using 50 µL 0.2% crystal violet (in PBS) add to 100 µL BAL(following other staining) for 1 min, and then wash off with PBS will reduce auto fluorescence considerably recommended by Dr Ruedi in Canada, thanks to you all please find below all the emails I received 1-Hello Ibtissman, You may want to switch to a larger nozzle if you have one. The nozzle diameter should be at least 3x the maximum cell size or else the cells may be damaged. If you're not sorting it's less of an issue, but I'd go larger if possible. Does propidium iodide go into protoplasts just like mammalian cells? You can read that in an "FL3" channel (either a 610 or 630nm bandpass filter) excited by 488 laser and I don't think you'll have trouble with spectral overlap with GFP. If so, you can try 7-AAD. Good luck, Peter Peter Lopez The Aaron Diamond AIDS Research Center 212.448.5188 (office) 2- iaj, B-D sells "FACS GFP Calibration Beads" Cat # 8375-1 which may be of use to you. The set up a standard curve that relates fluorescence intensity to MESF (Molecules of Equivalent Soluble Fluorochrome) of EGFP. (a cell with an MESF value of 5,000 is as bright as 5,000 EGFP molecules) If you generate this curve, and calculate the MESF value of the "negative" cells, you will have the autofluorescence of the cells as a quantitative value. This MESF value can then be subtracted from each of the positive cells to obtain the # of EGFP molecules they are labeled with. The beads are about 7 microns, but you can change your FSC and SSC parameters as you see fit in order to analyze the beads and then your cells, so long as yoiu do not adjust the fluorescence PMT's. If you need more info, give me a call! Best regards, -Nathan Nathan D. Foushee, MT (ASCP) Product Manager - Flow Cytometry Give us a call- Let's MultiPlex! www.QuantumPlex.com Bangs Laboratories, Inc. 9025 Technology Drive Fishers, IN 46038-2886 800.387.0672 317.570.7020 fax 317.570.7034 nathan@bangslabs.com www.bangslabs.com 3-have you tried autofluorescence compensation ?: Alberti S, Parks DR, Herzenberg LA.A single laser method for subtraction of cell autofluorescence in flow cytometry. Cytometry. 1987 Mar;8(2):114-9. Roederer M, Murphy RF.Cell-by-cell autofluorescence correction for low signal-to-noise systems: application to epidermal growth factor endocytosis by 3T3 fibroblasts. Cytometry. 1986 Nov;7(6):558-65. do you know the transfection efficiency of the protoplasts ? and why are there 2 populations that differ in FL1? Best wishes Rachel 4-You should use a nozzle of 120 or 150 um for this size of cells! A method I got from the mailing list for treating BAL: Quenching autofluorescence We have found adding 50 µL 0.2% crystal violet (in PBS) to 100 µL BAL (following other staining) for 1 min, and then wash off with PBS will reduce autofluorescence considerably Hope this helps Ruedi Dr. Ruedi K Braun Associated Scientist Ottawa Health Research Institute 501 Smyth Road Ottawa, ON K1H 8L6 Canada How about using DsRed2? It's as bright as eGFP, less toxic than DsRed, and emits where there's far less autofluorescence. <<...OLE_Obj...>> Andrew Andrew Beernink Research Scientist Manager, Flow Cytometry Novasite Pharmaceuticals, Inc. 11095 Flintkote San Diego, CA 92121 (858) 638-8584 5- Hello, We routinely run GFP vs Auto-Orange (PE filter). Here the Autofluorescent cells fall on the diagonal and the true GFP + cells are just off the diagonal. I've attached a file to demonstrate what I mean. 30u cells may be too large for a 70u nozzle. You might notice alot of spraying of the side streams when you try to sort because the large cells will perturb the droplet formation. I think the rule of thumb is the cell diameter should be about 1/4-1/5 the size of the nozzle orafice. Good Luck. Feel free to contact me if you have any questions. Lucy Lucy Brown Analytical Cytometry Core Beckman Research Institute/City of Hope Medical Center Duarte, California 626-359-8111 X63306 lbrown@coh.org 6- Hi, As an unrelated matter. Have you had any problem with clogs or abberant signals as a result of running such a large particle through the 70u Tip. I think most would agree that the stream flow would be much more laminar if the 100U Tip (which Cytomation should have provided at least one of when you got your instument) were used. If you ever sort these I almost sure that you will experience a lot of fanning of side streams with the 70u tip. A rule of thumb I learned a long time ago speaks to limiting the event size to 25% of the nozzle diameter especially for sorting. Some people push this. Cytomation also sells a 150u Tip used a lot by the marine biology& plant prototplast people. Sorry I had no answers for your primary question. Do you know of Galbraith at Univ. of Arizona? All he works with are plant cells and has presented alot of GFP/YFP/...FP work in the past. Regards, Don 7- iaj, What you show looks less like cells -- more like just "stuff" . . . First, using a 70 um tip for probable->30 um cells is a mistake. You need minimally a 3-4/1 ratio orifice size to cell size . . . 4-5/1 is better. Look at a >100 (120, 150, 200) um tip. Then . . . Second, assuming you can resolve cells, consider a different fluorescent protein (like DsRed) -- far less autofluoescence (if that's truly the problem). Finally . . . the anti-GFP ab will only work with a surface-associated GFP (unless, of course, you fix and permeabilise the cells to target intracellular GFP . . . but, that's a completely new set of perils . . .) MAK. 8-Iaj, We have found that if you acquire cells on a LogFL1(GFP) vs LogFL2 dotplot and set the instrument settings, so that the cells fall in the first decade, you can discriminate gfp +ve cells from background fluorescence by applying some FL2-FL1 compensation. This should bring any gfp +ves down towards the FL1 axis. If you keep the PMT settings for F1 and FL2 roughly equal you probably find you may need to apply about 15% compensation. This works well with cells but I can't say for protoplasts. Good luck. Regards, Jeff Barry Flow Cytometry Paterson Institute for Cancer Research Manchester UK 9- I am completely unfamiliar with plant protoblasts. First of all I would try log/log scatter plots in the hope to find some cell clustering by scatter. I take it you've identified their position in the light scatter plot by a DNA signal initially and ensured no DNA stain to remain in the system. Autofluorescence is volume dependent. Therefore if you look at log scatter vs log flr the cells should give you a diagonal and I would expect some "leaning over" of the cluster in the FSC / FL1 plot. Good luck and stay metric 10- Gerhard I have alot of experience at looking in transfected tobbacco and Zinnia protoplasts with green red and cyan fluorescent proteins. The plots you enclosed are a little unclear as to what gates are applied to what, if any of the plots. It looks like to me that you are excluding most of your protoplasts by thresholding on SSC-your gains appear too low relative to your thresholding channel. Regarding your autofluorescence problem- 11-How are you defining this?-autofluorescence is in part, Hi, As an unrelated matter. Have you had any problem with clogs or abberant signals as a result of running such a large particle through the 70u Tip. I think most would agree that the stream flow would be much more laminar if the 100U Tip (which Cytomation should have provided at least one of when you got your instument) were used. If you ever sort these I almost sure that you will experience a lot of fanning of side streams with the 70u tip. A rule of thumb I learned a long time ago speaks to limiting the event size to 25% of the nozzle diameter especially for sorting. Some people push this. Cytomation also sells a 150u Tip used a lot by the marine biology& plant prototplast people. Sorry I had no answers for your primary question. Do you know of Galbraith at Univ. of Arizona? All he works with are plant cells and has presented alot of GFP/YFP/...FP work in the past. Regards, Don 12- I have not personally ever had a problem in distinguishing between background FL1 and GFP+ fluorescence in protoplasts, once the non viable cells have been excluded from analytical plot. You should drop your FL1 gains abit and bring your background fluorescence down to 1st decade. You will actually gain more visual sensitivity for GFP Dim cells if you do this. c2189
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