Stopping Mitochondrial Activity in whole mount organs
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Romin, Yevgeniy/Sloan Kettering Institute |
Stopping Mitochondrial Activity in whole mount organs
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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 everybody. This isn't exactly a microscopy question, but maybe someone here has experience with this issue. We are doing experiments involving assessing the viability of cells in whole mount live organs (liver, in this case), and we are using Mitotracker in order to see whether the cells are alive. Lately we have been doubting the integrity of our Mitotracker staining, and we would like a negative control to work with. Does anybody have any experience with stopping the mitochondrial activity in live tissues, like anything that we could inject into the organ? We need these negative controls to be on tissue, not on cells. Any advice you can give will be greatly appreciated. --------------------------------------------------- Yevgeniy Romin Digital Microscopist Memorial Sloan-Kettering Cancer Center Molecular Cytology Core Facility 1275 York Ave. Box 333 New York, NY 10065 Tel.646-888-2186 Fax. 646-422-0640 --------------------------------------------------- ===================================================================== Please note that this e-mail and any files transmitted with it may be privileged, confidential, and protected from disclosure under applicable law. If the reader of this message is not the intended recipient, or an employee or agent responsible for delivering this message to the intended recipient, you are hereby notified that any reading, dissemination, distribution, copying, or other use of this communication or any of its attachments is strictly prohibited. If you have received this communication in error, please notify the sender immediately by replying to this message and deleting this message, any attachments, and all copies and backups from your computer. |
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Z.J. Zhang |
Re: Stopping Mitochondrial Activity in whole mount organs
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Did you try Potassium cyanide (KCN), which would stop the oxidative phosphorylation of mitochondria? Zhaojie Zhaojie Zhang, Ph. D. Director, Jenkins Microscopy Facility University of Wyoming Laramie, WY 82071 PHONE: 307-766-3038 FAX: 307-766-5625 -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Yevgeniy Romin Sent: Tuesday, January 18, 2011 8:51 AM To: [hidden email] Subject: Stopping Mitochondrial Activity in whole mount organs ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hello everybody. This isn't exactly a microscopy question, but maybe someone here has experience with this issue. We are doing experiments involving assessing the viability of cells in whole mount live organs (liver, in this case), and we are using Mitotracker in order to see whether the cells are alive. Lately we have been doubting the integrity of our Mitotracker staining, and we would like a negative control to work with. Does anybody have any experience with stopping the mitochondrial activity in live tissues, like anything that we could inject into the organ? We need these negative controls to be on tissue, not on cells. Any advice you can give will be greatly appreciated. --------------------------------------------------- Yevgeniy Romin Digital Microscopist Memorial Sloan-Kettering Cancer Center Molecular Cytology Core Facility 1275 York Ave. Box 333 New York, NY 10065 Tel.646-888-2186 Fax. 646-422-0640 --------------------------------------------------- ===================================================================== Please note that this e-mail and any files transmitted with it may be privileged, confidential, and protected from disclosure under applicable law. If the reader of this message is not the intended recipient, or an employee or agent responsible for delivering this message to the intended recipient, you are hereby notified that any reading, dissemination, distribution, copying, or other use of this communication or any of its attachments is strictly prohibited. If you have received this communication in error, please notify the sender immediately by replying to this message and deleting this message, any attachments, and all copies and backups from your computer. |
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Boswell, Carl A - (cboswell) |
Re: Stopping Mitochondrial Activity in whole mount organs
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In reply to this post by Romin, Yevgeniy/Sloan Kettering Institute
Because tissue function depends on cell function that makes up the tissue, and because mitochondria are intracellular, treating tissue without treating cells is not possible. Moreover, poisoning mitochondria with cyanide, which will certainly stop their function, will quickly kill the cells/tissue, so tissue function would stop. Because the mitochondrion is so fundamental to the workings of a cell, I don't see a way out. I'm trying to imagine a fully functional eukaryotic cell without mitochondria. You could certainly treat with cyanide, then see how Mitotracker stains, but the tissue will likely not be functional at the same time.
C Carl A. Boswell, Ph.D. Molecular and Cellular Biology Univ. of Arizona 520-954-7053 FAX 520-621-3709 -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Yevgeniy Romin Sent: Tuesday, January 18, 2011 8:51 AM To: [hidden email] Subject: Stopping Mitochondrial Activity in whole mount organs ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hello everybody. This isn't exactly a microscopy question, but maybe someone here has experience with this issue. We are doing experiments involving assessing the viability of cells in whole mount live organs (liver, in this case), and we are using Mitotracker in order to see whether the cells are alive. Lately we have been doubting the integrity of our Mitotracker staining, and we would like a negative control to work with. Does anybody have any experience with stopping the mitochondrial activity in live tissues, like anything that we could inject into the organ? We need these negative controls to be on tissue, not on cells. Any advice you can give will be greatly appreciated. --------------------------------------------------- Yevgeniy Romin Digital Microscopist Memorial Sloan-Kettering Cancer Center Molecular Cytology Core Facility 1275 York Ave. Box 333 New York, NY 10065 Tel.646-888-2186 Fax. 646-422-0640 --------------------------------------------------- ===================================================================== Please note that this e-mail and any files transmitted with it may be privileged, confidential, and protected from disclosure under applicable law. If the reader of this message is not the intended recipient, or an employee or agent responsible for delivering this message to the intended recipient, you are hereby notified that any reading, dissemination, distribution, copying, or other use of this communication or any of its attachments is strictly prohibited. If you have received this communication in error, please notify the sender immediately by replying to this message and deleting this message, any attachments, and all copies and backups from your computer. |
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Tobias Baskin |
Re: Stopping Mitochondrial Activity in whole mount organs
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Greetings, While the tenor of Carl's remarks is right, keep in mind that tissues differ in their ability to live aneorobically. Some tissue/organs can survive hours without oxidative phosphorylation. Of course this will induce various changes in the cell physiology but these can stop short of death. I don't know about KCN but there are certain uncouplers (e.g., dinitrophenol) that will stop oxphos without directly affecting other componenets. I think oligomycin is another. I expect that membrane potential would decrease within minutes so add mitotracker at that point and see. Not the greatest experiment in the world but could serve your purpose. As ever Tobias Baskin >Because tissue function depends on cell function that makes up the >tissue, and because mitochondria are intracellular, treating tissue >without treating cells is not possible. Moreover, poisoning >mitochondria with cyanide, which will certainly stop their function, >will quickly kill the cells/tissue, so tissue function would stop. >Because the mitochondrion is so fundamental to the workings of a >cell, I don't see a way out. I'm trying to imagine a fully >functional eukaryotic cell without mitochondria. You could >certainly treat with cyanide, then see how Mitotracker stains, but >the tissue will likely not be functional at the same time. > >C > >Carl A. Boswell, Ph.D. >Molecular and Cellular Biology >Univ. of Arizona >520-954-7053 >FAX 520-621-3709 > > >-----Original Message----- >From: Confocal Microscopy List >[mailto:[hidden email]] On Behalf Of Yevgeniy Romin >Sent: Tuesday, January 18, 2011 8:51 AM >To: [hidden email] >Subject: Stopping Mitochondrial Activity in whole mount organs > >***** >To join, leave or search the confocal microscopy listserv, go to: >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >***** > >Hello everybody. This isn't exactly a microscopy question, but >maybe someone here has experience with this issue. > >We are doing experiments involving assessing the viability of cells >in whole mount live organs (liver, in this case), and we are using >Mitotracker in order to see whether the cells are alive. Lately we >have been doubting the integrity of our Mitotracker staining, and we >would like a negative control to work with. Does anybody have any >experience with stopping the mitochondrial activity in live tissues, >like anything that we could inject into the organ? We need these >negative controls to be on tissue, not on cells. Any advice you can >give will be greatly appreciated. > >--------------------------------------------------- >Yevgeniy Romin > >Digital Microscopist >Memorial Sloan-Kettering Cancer Center >Molecular Cytology Core Facility >1275 York Ave. Box 333 >New York, NY 10065 >Tel.646-888-2186 >Fax. 646-422-0640 >--------------------------------------------------- > > > > ===================================================================== > > Please note that this e-mail and any files transmitted with it may be > privileged, confidential, and protected from disclosure under > applicable law. If the reader of this message is not the intended > recipient, or an employee or agent responsible for delivering this > message to the intended recipient, you are hereby notified that any > reading, dissemination, distribution, copying, or other use of this > communication or any of its attachments is strictly prohibited. If > you have received this communication in error, please notify the > sender immediately by replying to this message and deleting this > message, any attachments, and all copies and backups from your > computer. |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Yevgeniy, Uncouplers like CCCP, FCCP, DNP can block mitochondrial ATP synthesis by collapsing the proton gradient, but as Tobias and Carl point out, many intracellular processes will quickly come to a halt as ATP concentrations are consumed. Choosing a cell type that has a high membrane impermeability, reducing the necessity of ATP pumps, and supports a functional glycolytic pathway would seem to be crucial. You might be able to drop the temperature to slow down metabolism as well. To obtain meaningful data it would seem that you should monitor a number of physiological parameters simultaneously including cell volume, intracellular Ca++, PM potential, etc Mario >***** >To join, leave or search the confocal microscopy listserv, go to: >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >***** > >Greetings, > While the tenor of Carl's remarks is right, keep in mind that >tissues differ in their ability to live aneorobically. Some >tissue/organs can survive hours without oxidative phosphorylation. >Of course this will induce various changes in the cell physiology >but these can stop short of death. I don't know about KCN but there >are certain uncouplers (e.g., dinitrophenol) that will stop oxphos >without directly affecting other componenets. I think oligomycin is >another. I expect that membrane potential would decrease within >minutes so add mitotracker at that point and see. Not the greatest >experiment in the world but could serve your purpose. > > As ever > Tobias Baskin > >>Because tissue function depends on cell function that makes up the >>tissue, and because mitochondria are intracellular, treating tissue >>without treating cells is not possible. Moreover, poisoning >>mitochondria with cyanide, which will certainly stop their >>function, will quickly kill the cells/tissue, so tissue function >>would stop. Because the mitochondrion is so fundamental to the >>workings of a cell, I don't see a way out. I'm trying to imagine a >>fully functional eukaryotic cell without mitochondria. You could >>certainly treat with cyanide, then see how Mitotracker stains, but >>the tissue will likely not be functional at the same time. >> >>C >> >>Carl A. Boswell, Ph.D. >>Molecular and Cellular Biology >>Univ. of Arizona >>520-954-7053 >>FAX 520-621-3709 >> >> >>-----Original Message----- >>From: Confocal Microscopy List >>[mailto:[hidden email]] On Behalf Of Yevgeniy >>Romin >>Sent: Tuesday, January 18, 2011 8:51 AM >>To: [hidden email] >>Subject: Stopping Mitochondrial Activity in whole mount organs >> >>***** >>To join, leave or search the confocal microscopy listserv, go to: >>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>***** >> >>Hello everybody. This isn't exactly a microscopy question, but >>maybe someone here has experience with this issue. >> >>We are doing experiments involving assessing the viability of cells >>in whole mount live organs (liver, in this case), and we are using >>Mitotracker in order to see whether the cells are alive. Lately we >>have been doubting the integrity of our Mitotracker staining, and >>we would like a negative control to work with. Does anybody have >>any experience with stopping the mitochondrial activity in live >>tissues, like anything that we could inject into the organ? We >>need these negative controls to be on tissue, not on cells. Any >>advice you can give will be greatly appreciated. >> >>--------------------------------------------------- >>Yevgeniy Romin >> >>Digital Microscopist >>Memorial Sloan-Kettering Cancer Center >>Molecular Cytology Core Facility >>1275 York Ave. Box 333 >>New York, NY 10065 >>Tel.646-888-2186 >>Fax. 646-422-0640 >>--------------------------------------------------- >> >> >> >> ===================================================================== >> >> Please note that this e-mail and any files transmitted with it may be >> privileged, confidential, and protected from disclosure under >> applicable law. If the reader of this message is not the intended >> recipient, or an employee or agent responsible for delivering this >> message to the intended recipient, you are hereby notified that any >> reading, dissemination, distribution, copying, or other use of this >> communication or any of its attachments is strictly prohibited. If >> you have received this communication in error, please notify the >> sender immediately by replying to this message and deleting this >> message, any attachments, and all copies and backups from your >> computer. -- ________________________________________________________________________________ Mario M. Moronne, Ph.D. [hidden email] [hidden email] |
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Romin, Yevgeniy/Sloan Kettering Institute |
Re: Stopping Mitochondrial Activity in whole mount organs
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In reply to this post by Tobias Baskin
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Thank you all very much for your suggestions, we will give it a try. --------------------------------------------------- Yevgeniy Romin Digital Microscopist Memorial Sloan-Kettering Cancer Center Molecular Cytology Core Facility 1275 York Ave. Box 333 New York, NY 10065 Tel.646-888-2186 Fax. 646-422-0640 --------------------------------------------------- -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Tobias Baskin Sent: Tuesday, January 18, 2011 1:40 PM To: [hidden email] Subject: Re: Stopping Mitochondrial Activity in whole mount organs ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Greetings, While the tenor of Carl's remarks is right, keep in mind that tissues differ in their ability to live aneorobically. Some tissue/organs can survive hours without oxidative phosphorylation. Of course this will induce various changes in the cell physiology but these can stop short of death. I don't know about KCN but there are certain uncouplers (e.g., dinitrophenol) that will stop oxphos without directly affecting other componenets. I think oligomycin is another. I expect that membrane potential would decrease within minutes so add mitotracker at that point and see. Not the greatest experiment in the world but could serve your purpose. As ever Tobias Baskin >Because tissue function depends on cell function that makes up the >tissue, and because mitochondria are intracellular, treating tissue >without treating cells is not possible. Moreover, poisoning >mitochondria with cyanide, which will certainly stop their function, >will quickly kill the cells/tissue, so tissue function would stop. >Because the mitochondrion is so fundamental to the workings of a >cell, I don't see a way out. I'm trying to imagine a fully >functional eukaryotic cell without mitochondria. You could >certainly treat with cyanide, then see how Mitotracker stains, but >the tissue will likely not be functional at the same time. > >C > >Carl A. Boswell, Ph.D. >Molecular and Cellular Biology >Univ. of Arizona >520-954-7053 >FAX 520-621-3709 > > >-----Original Message----- >From: Confocal Microscopy List >[mailto:[hidden email]] On Behalf Of Yevgeniy Romin >Sent: Tuesday, January 18, 2011 8:51 AM >To: [hidden email] >Subject: Stopping Mitochondrial Activity in whole mount organs > >***** >To join, leave or search the confocal microscopy listserv, go to: >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >***** > >Hello everybody. This isn't exactly a microscopy question, but >maybe someone here has experience with this issue. > >We are doing experiments involving assessing the viability of cells >in whole mount live organs (liver, in this case), and we are using >Mitotracker in order to see whether the cells are alive. Lately we >have been doubting the integrity of our Mitotracker staining, and we >would like a negative control to work with. Does anybody have any >experience with stopping the mitochondrial activity in live tissues, >like anything that we could inject into the organ? We need these >negative controls to be on tissue, not on cells. Any advice you can >give will be greatly appreciated. > >--------------------------------------------------- >Yevgeniy Romin > >Digital Microscopist >Memorial Sloan-Kettering Cancer Center >Molecular Cytology Core Facility >1275 York Ave. Box 333 >New York, NY 10065 >Tel.646-888-2186 >Fax. 646-422-0640 >--------------------------------------------------- > > > > ===================================================================== > > Please note that this e-mail and any files transmitted with it may be > privileged, confidential, and protected from disclosure under > applicable law. If the reader of this message is not the intended > recipient, or an employee or agent responsible for delivering this > message to the intended recipient, you are hereby notified that any > reading, dissemination, distribution, copying, or other use of this > communication or any of its attachments is strictly prohibited. If > you have received this communication in error, please notify the > sender immediately by replying to this message and deleting this > message, any attachments, and all copies and backups from your > computer. |
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