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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hello List, I wonder if anybody has a positive experience with Fura -2 Ca Imaging calibration kit (F-6774) from Molecular probes. My problem is that fluorescence signals are very weak (much weaker than for Fura-2 stained cells) and do not produce a linear calibration curve. I wonder if this may be related to the number of beads on the slides. In most slides I got very few beads (<10). Maybe this is not enough to provide uniform thickness of the fluorescent solution. I vortexed the buffers with beads before preparing slides but after some time (~10 min) most likely the beads settled because the last slides had much fewer (or none) beads. In some first slides there are many beads stuck together. Molecular probes provides a very detail protocol. However, they do not tell what density of beads should be observed on the prepared slides. Theoretical calculations show that for 16,000/mL bead concentration (0.0028%) the beads would be spaced in 15um layer by ~2mm on average. In my slides wherever I see the beads they seem to be more densely packed. To measure Ca in my solution, I added 15um beads from polyscience (original conc. 1%) diluted in final solution 300 times. However, again got very few beads on the slide. Any feedback and/or advice is very much appreciated. Thanks, Arvydas ******************** Arvydas Matiukas, Ph.D. Director of Confocal&Two-Photon Core Department of Pharmacology SUNY Upstate Medical University 766 Irving Ave., WH 3167 Syracuse, NY 13210 tel.: 315-464-7997 fax: 315-464-8014 email: [hidden email] |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** What concentration of fura-2 in the calibration solution are you using? When you say a linear calibration curve what do you mean -the dye is not linear but shows saturation as well as a non-calcium dependent component. For the calirbartion to have validity, you must use the same equipment exactly as was/is used to image the cells -what wavelengths are you using? Cheers Mark On 5/12/2011, at 8:40 PM, Arvydas Matiukas wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hello List, > > I wonder if anybody has a positive experience with Fura -2 Ca Imaging calibration kit > (F-6774) from Molecular probes. > > My problem is that fluorescence signals are very weak (much weaker than for > Fura-2 stained cells) and do not produce a linear calibration curve. I wonder if this may be > related to the number of beads on the slides. > > In most slides I got very few beads (<10). Maybe this is not enough to provide > uniform thickness of the fluorescent solution. I vortexed the buffers with beads before preparing slides > but after some time (~10 min) most likely the beads settled because the last slides > had much fewer (or none) beads. In some first slides there are many beads stuck together. > > Molecular probes provides a very detail protocol. However, they do not tell what density > of beads should be observed on the prepared slides. Theoretical calculations > show that for 16,000/mL bead concentration (0.0028%) the beads would be spaced in 15um > layer by ~2mm on average. In my slides wherever I see the beads they seem to be more > densely packed. > To measure Ca in my solution, I added 15um beads from polyscience (original conc. 1%) diluted in > final solution 300 times. However, again got very few beads on the slide. > > Any feedback and/or advice is very much appreciated. > > Thanks, > Arvydas > ******************** > > > Arvydas Matiukas, Ph.D. > Director of Confocal&Two-Photon Core > Department of Pharmacology > SUNY Upstate Medical University > 766 Irving Ave., WH 3167 > Syracuse, NY 13210 > tel.: 315-464-7997 > fax: 315-464-8014 > email: [hidden email] |
Arvydas Matiukas |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Mark, I followed the protocol provided by Molecular probes for the kit F-6774 . Fura-2 concentration in the calibration solutions is 50uM. I used the same microscope to image cells and the calibration solutions at excitation wavelengths 340 and 380 nm. By nonlinearity I mean that log plot of F340/F380 ratio (background corrected ) is strongly nonlinear vs log Ca concentration. What bothers me most is that the fluorescence signals (background corrected) from the calibration solutions with 50uM Fura-2 are much weaker ( 10- 150 units) than from 5 uM Fura-2 AM stained N2A cells (100- 1500 units). Any feedback/comments by the list are greatly appreciated. Thanks, Arvydas >>> Mark Cannell <[hidden email]> 12/6/2011 4:56 AM >>> ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** What concentration of fura-2 in the calibration solution are you using? When you say a linear calibration curve what do you mean -the dye is not linear but shows saturation as well as a non-calcium dependent component. For the calirbartion to have validity, you must use the same equipment exactly as was/is used to image the cells -what wavelengths are you using? Cheers Mark On 5/12/2011, at 8:40 PM, Arvydas Matiukas wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hello List, > > I wonder if anybody has a positive experience with Fura -2 Ca Imaging calibration kit > (F-6774) from Molecular probes. > > My problem is that fluorescence signals are very weak (much weaker than for > Fura-2 stained cells) and do not produce a linear calibration curve. I wonder if this may be > related to the number of beads on the slides. > > In most slides I got very few beads (<10). Maybe this is not enough to provide > uniform thickness of the fluorescent solution. I vortexed the buffers with beads before preparing slides > but after some time (~10 min) most likely the beads settled because the last slides > had much fewer (or none) beads. In some first slides there are many beads stuck together. > > Molecular probes provides a very detail protocol. However, they do not tell what density > of beads should be observed on the prepared slides. Theoretical calculations > show that for 16,000/mL bead concentration (0.0028%) the beads would be spaced in 15um > layer by ~2mm on average. In my slides wherever I see the beads they seem to be more > densely packed. > To measure Ca in my solution, I added 15um beads from polyscience (original conc. 1%) diluted in > final solution 300 times. However, again got very few beads on the slide. > > Any feedback and/or advice is very much appreciated. > > Thanks, > Arvydas > ******************** > > > Arvydas Matiukas, Ph.D. > Director of Confocal&Two-Photon Core > Department of Pharmacology > SUNY Upstate Medical University > 766 Irving Ave., WH 3167 > Syracuse, NY 13210 > tel.: 315-464-7997 > fax: 315-464-8014 > email: [hidden email] |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Well, the background should be higher in cells due to autofluorescence -right? Second a log-log plot will only be linear with a slope of 1 well below the Kd. Even at 0.1 Kd, the slope will be 0.85 decreasing to 0.25 at the Kd. Is that what you are seeing? Hope this helps Mark On 6/12/2011, at 3:52 PM, Arvydas Matiukas wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hi Mark, > > I followed the protocol provided by Molecular probes for the kit F-6774 . Fura-2 concentration > in the calibration solutions is 50uM. I used the same microscope to image cells and the > calibration solutions at excitation wavelengths 340 and 380 nm. By nonlinearity I mean > that log plot of F340/F380 ratio (background corrected ) is strongly nonlinear vs log Ca concentration. > > What bothers me most is that the fluorescence signals (background corrected) from > the calibration solutions with 50uM Fura-2 are much weaker ( 10- 150 units) than > from 5 uM Fura-2 AM stained N2A cells (100- 1500 units). > > Any feedback/comments by the list are greatly appreciated. > > Thanks, > Arvydas > > > > >>>> Mark Cannell <[hidden email]> 12/6/2011 4:56 AM >>> > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > What concentration of fura-2 in the calibration solution are you using? When you say a linear calibration curve what do you mean -the dye is not linear but shows saturation as well as a non-calcium dependent component. For the calirbartion to have validity, you must use the same equipment exactly as was/is used to image the cells -what wavelengths are you using? > > Cheers Mark > > On 5/12/2011, at 8:40 PM, Arvydas Matiukas wrote: > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> Hello List, >> >> I wonder if anybody has a positive experience with Fura -2 Ca Imaging calibration kit >> (F-6774) from Molecular probes. >> >> My problem is that fluorescence signals are very weak (much weaker than for >> Fura-2 stained cells) and do not produce a linear calibration curve. I wonder if this may be >> related to the number of beads on the slides. >> >> In most slides I got very few beads (<10). Maybe this is not enough to provide >> uniform thickness of the fluorescent solution. I vortexed the buffers with beads before preparing slides >> but after some time (~10 min) most likely the beads settled because the last slides >> had much fewer (or none) beads. In some first slides there are many beads stuck together. >> >> Molecular probes provides a very detail protocol. However, they do not tell what density >> of beads should be observed on the prepared slides. Theoretical calculations >> show that for 16,000/mL bead concentration (0.0028%) the beads would be spaced in 15um >> layer by ~2mm on average. In my slides wherever I see the beads they seem to be more >> densely packed. >> To measure Ca in my solution, I added 15um beads from polyscience (original conc. 1%) diluted in >> final solution 300 times. However, again got very few beads on the slide. >> >> Any feedback and/or advice is very much appreciated. >> >> Thanks, >> Arvydas >> ******************** >> >> >> Arvydas Matiukas, Ph.D. >> Director of Confocal&Two-Photon Core >> Department of Pharmacology >> SUNY Upstate Medical University >> 766 Irving Ave., WH 3167 >> Syracuse, NY 13210 >> tel.: 315-464-7997 >> fax: 315-464-8014 >> email: [hidden email] |
Boswell, Carl A - (cboswell) |
In reply to this post by Arvydas Matiukas
Hi Arvydas,
It's been many years since I did Ca measurements with Fura-2, well before the beads were available. However I remember that the intracellular, and often, subcellular concentration of Fura-2 was well beyond what the bathing solution was. This was due to the disequilibrium between the nearly constant concentration of the AM version of the dye outside the cell and the increasing concentration of Fura-2 trapped inside once the AM moiety was removed by intracellular esterases. With poorly monitored loading technique, one could get nearly mM Fura inside cells. The bottom line is possibly that you have a much higher intracellular concentration of Fura-2 inside your loaded cells than in your calibration solution, hence the brighter signal. You should also appreciate the sensitivity of Fura-2 Kd to pH, and how little control, or knowledge, you have of that parameter inside the cell. As a consequence, extrapolating values from a calibration solution to intracellular reality should be done cautiously. Good luck, C Carl A. Boswell 520-954-7053 -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Arvydas Matiukas Sent: Tuesday, December 06, 2011 8:53 AM To: [hidden email] Subject: Re: Ca Imaging calibration kit ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Mark, I followed the protocol provided by Molecular probes for the kit F-6774 . Fura-2 concentration in the calibration solutions is 50uM. I used the same microscope to image cells and the calibration solutions at excitation wavelengths 340 and 380 nm. By nonlinearity I mean that log plot of F340/F380 ratio (background corrected ) is strongly nonlinear vs log Ca concentration. What bothers me most is that the fluorescence signals (background corrected) from the calibration solutions with 50uM Fura-2 are much weaker ( 10- 150 units) than from 5 uM Fura-2 AM stained N2A cells (100- 1500 units). Any feedback/comments by the list are greatly appreciated. Thanks, Arvydas >>> Mark Cannell <[hidden email]> 12/6/2011 4:56 AM >>> ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** What concentration of fura-2 in the calibration solution are you using? When you say a linear calibration curve what do you mean -the dye is not linear but shows saturation as well as a non-calcium dependent component. For the calirbartion to have validity, you must use the same equipment exactly as was/is used to image the cells -what wavelengths are you using? Cheers Mark On 5/12/2011, at 8:40 PM, Arvydas Matiukas wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hello List, > > I wonder if anybody has a positive experience with Fura -2 Ca Imaging > calibration kit > (F-6774) from Molecular probes. > > My problem is that fluorescence signals are very weak (much weaker > than for > Fura-2 stained cells) and do not produce a linear calibration curve. I > wonder if this may be related to the number of beads on the slides. > > In most slides I got very few beads (<10). Maybe this is not enough to > provide uniform thickness of the fluorescent solution. I vortexed > the buffers with beads before preparing slides but after some time > (~10 min) most likely the beads settled because the last slides had much fewer (or none) beads. In some first slides there are many beads stuck together. > > Molecular probes provides a very detail protocol. However, they do > not tell what density of beads should be observed on the prepared > slides. Theoretical calculations show that for 16,000/mL bead > concentration (0.0028%) the beads would be spaced in 15um layer by > ~2mm on average. In my slides wherever I see the beads they seem to be more densely packed. > To measure Ca in my solution, I added 15um beads from polyscience > (original conc. 1%) diluted in final solution 300 times. However, again got very few beads on the slide. > > Any feedback and/or advice is very much appreciated. > > Thanks, > Arvydas > ******************** > > > Arvydas Matiukas, Ph.D. > Director of Confocal&Two-Photon Core > Department of Pharmacology > SUNY Upstate Medical University > 766 Irving Ave., WH 3167 > Syracuse, NY 13210 > tel.: 315-464-7997 > fax: 315-464-8014 > email: [hidden email] |
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