From: Nebe-Von-Caron, G (g.nebe-von-caron@unipath.com)
Date: Tue Aug 26 2003 - 07:11:00 EST
Regarding the lack of impact on sheath buffers on sample measurement I would be careful about 2 things here. >From recent work on the calibur I have found that there is quite a bit of liquid transferred from the outer tube that is supposed to suck up the drops that come back from the sample tube leading to the flow cell. It definitely transferred enough cells and PI to cause us trouble. Thus it is worthwhile to suck that liquid into a tissue - or get the instrument fixed if that is a fault condition. Whilst indeed the sample buffer is more important to cell physiology than the sheath, the contact time between cells and surrounding sheath is longer in a flow cell based system compared to a jet in air system due to the lower velocity inside the flow cell. If you read up about the H-filter micro fluidics one can get the idea of how nicely molecules can move across those 'laminar flow boundarys'. So depending on the sheath composition and the relative volumes of sheath to sample flow you will get significantly different results. This is most important when trying to do intracellular pH measurements as protons have a very high diffusion coefficient. I used covalently bound carboxyfluorescein-diacetate-succinimidylester tried to obtain calibration measurements in bacteria. My initial attempts to use a 'plug-flow-sheath' approach failed as the plug quite quickly assumed the normal pH of the sheath. You can only calibrate your fluorescent measurements if your sheath fluid is neutral and unbuffered. I eventually used 1/10th strength saline as sheath that turned out fairly neutral but goes acidic over time due to dissolved CO2. The resulting sheath / sample mix measured with a 60ul pH probe (pH-boy http://www.camlab.co.uk/pages/product.asp?catid=968 <http://www.camlab.co.uk/pages/product.asp?catid=968&sid=3f4b4d7b036cb81a273fc16ca96906eb&custno=WWWanonymous> &sid=3f4b4d7b036cb81a273fc16ca96906eb&custno=WWWanonymous ) turned out to be very close to the desired pH. Other effects are obviously refractive index match... Regarding the use of DI sheath it is just that your cells can withstand the osmotic pressure for some time. This dynamic principle is actually used as a lysis procedure in the Abbot Cell-dyn haematology analysers. I hope Howard of Volker Kachel who did quite a lot on fluidics or someone else can elaborate a bit further on this issue All the best Gerhard Nebe-von-Caron Research Scientist and Biomedical Engineer Unipath Ltd Priory Business Park Bedford MK44 3UP Tel +44(0)1234-835474 Fax +44(0)1234-835002 -----Original Message----- From: Ronald Rabin [mailto:rr84g@nih.gov] Sent: 14 August 2003 16:54 To: cyto-inbox Subject: Re: Fluo-3 loading in Platelets Simone, how broad is your baseline with the fluo-3? The broader your baseline, the more noise you have, and the less sensitivity you have. Generally you want a linear scale because the dynamic range of calcium responses is not that wide. The sheath fluid is not as important as what the cells are suspended in. The cells don't see the sheath fluid because of the laminar flow. (I once ran an experiment accidently using DI rather than saline as sheath fluid and didn't catch it until I let the sheath (DI) back flush into the sample at which point they all showed a flux). So the buffer in which the platelets are suspended must be physiologic with respect to calcium concentration. The first thing to do is to use ionomycin. In a physiologic buffer, you should see a rise in the ratio by a factor of about six (true for lymphs, anyway). If you don't, your problem is technical. If you do, then the issue is biology/sensitivity. Once you are certain about your biology (see below) you will have to titer your indicator, as too much can buffer the calcium changes. You need to do that with your physiologic system, not ionomycin. (Kevin Holmes is laughing if he is reading this part because I ignored this advice from him for a while. . .) I don't know much about platelets. Obviously, they lack nuclei. Do they have ER? Does the ER function as calcium storage? What is the phenomenon you are looking for? Is it a G-protein-like phenomena where there is release from intracellular stores? Is it movement across the plasma membrane? If it is the former, the flux may not be of a magnitude that you can distinguish without ratiometric analysis. This can be done with two dyes (Fura-red is the other, I think) on the Calibur. If you are looking for movement of calcium across the cell membrane, the above paragraph is particularly relevant. Finally, I would suggest that you use either FL3 or FL4 for your CD marker. Linear/log compensation is a little tricky, and if you can avoid it by using a dye that does not overlap with FITC spectra, it is one less thing to deal with. ron On Tuesday, August 12, 2003, at 10:51 PM, SIMONE BOER wrote: Hi guys, This is an URGENT call out to anyone who may be able to help: I have been working for some time on a protocol (based on the whole blood method by do Ceu Monteiro, 1999) measuring platelet calcium levels in whole blood. The machine we are using is a FACSCalibur benchtop analyser by Becton Dickinson and we are using the calcium dye: Fluo-3 AM. This Flow Cytometer does not have UV fluorescence capacities and we do not have access to UV facilities at our location The following is our protocol: Blood is collected in vacutainer tubes with sodium citrate (to stop activation of the platelets) Blood is diluted 1:10 with modified Tyrodes buffer (containing 137mM NaCl, 2.8mM KCl, 1mM MgCl, 12mM NaHCO6, 0.4mM Na2HPO4, 0.35% BSA, 10mM HEPES, 5.5mM glucose, pH 7.4) and incubated with 5uM Fluo-3 AM (prepared from a 1mM stock solution in DMSO) for 15minutes at 37C in the dark Sample is then incubated with the platelet-labelling antibody CD41-PE for 15minutes, RT. At the end of incubation, modified Tyrodes buffer is added to the sample and data is collected, using Fl1 for Fluo-3 fluorescence and Fl2 for CD41 fluorescence. Changes in calcium fluorescence is plotted on a fluo-3 versus time graph After 20 seconds of baseline collection, an agonist (in this case - glutamate with concentrations ranging from 10nM to 1mM) is added to the sample Data is collected up to 3 minutes after agonist is added. This is a basic version of our protocol. Loading of the platelets with fluo-3 alone results in a relatively strong signal. However the problem is, that we do not get any increase in fluorescence reflecting calcium spikes, as a response to calcium when we add the agonist. We do not know if the problem is the platelets not responding or the fluo-3 not responding to shifts in calcium. We have tried several optimisation techniques and titrations to try and combat this problem: 1) We believed we may have "missed" the peak and tried to continuously collecting data. We even developed a syringe-like device that can add the agonist continuously without pausing the flow. This has not resulted in any calcium response being monitored. 2) We have added 2.5mM probenecid to incubation mixture to stop leakage of dyes. 3) We have incubated the fluo at RT to and tried adding the agonist. 4) In order to dismiss the idea that there was a problem with the glutamate, we have tried an alternative agonist, thrombin, (1U/mL) 5) We also tried to saturate the cell with glutamate to try and "force" any kind of response. We added glutamate at 1mM and we still get no response 6) We have even added Calcium Chloride (1mM) to the incubation mixture to incubate with the Fluo-3 , we have also added calcium to the sample 5 minutes before analysis and also tried adding calcium chloride during analysis 7) We have even added EGTA to the incubation mixture to chelate the extracellular calcium to change the membrane gradient 8) We have tried many differing concentrations of Fluo-3 and incubation times to try and produce a result 9) Data has been collected on both logarithmic and linear scales 10) Using the back-gating technique; we have stimulated platelets without CD41 present to exclude any aggregating effects of CD41, to no avail 11) The sheath fluid used is 0.9% isotonic saline None of the above results in any change in fluorescence Currently I am an honours student, working with an individual who has extemsive experience in FLow Cytometry. We have exhausted all possibilities and are desperate for any advice. With the honours year finishing in October, we are highly aware of the time constraints and with no results to date, we are becoming quite alarmed. Your replies are greatly appreciated Thanking everyone Simone Boer The University of Melbourne Barwon Health E-MAIL IS CONFIDENTIAL. If you have received this e-mail in error, please notify us by return e-mail and delete the document. If you are not the intended recipient you are hereby notified that any disclosure, copying, distribution or taking any action in reliance on the contents of this information is strictly prohibited and may be unlawful. Barwon Health is not liable for the proper and complete transmission of the information contained in this communication or for any delay in its receipt. Ronald L. Rabin, M.D. Senior Staff Fellow Laboratory of Immunobiochemistry DBPAP/OVRR Center for Biologics Evaluation and Research U.S. Food and Drug Administration 29 Lincoln Drive (MSC-4555) Building 29, Room 129 Bethesda, MD 20892-4555 phone: 301.496.8806 fax: 301.402.5177 email: rr84g@nih.gov
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