NetNotes


Bob Price University of South Carolina School of Medicine Bob.Price@uscmed.sc.edu


Analysis of Apoptosis Confocal Listserver We would like to analyze cell death over a 24h time lapse in cultured


cells that already express Venus. We would appreciate any recommendations for reagent that can last at least 24h with an EM/Ex spectrum that does not overlap with the spectrum of Venus. Any other advice is also very welcome. Tanks in advance. Verona Villar vvillar@umh.es


Not sure about 24h, but I think CellEvent is used for long


incubations. Mike Model mmodel@kent.edu I don’t know if this will be helpful but I’m linking to a video


(https://www.dropbox.com/s/okmxszk4vng3010/Dying%20cells. mp4?dl=0) showing a cell death experiment where we put calcein AM and propidium iodide (PI) in the media and also stained the cells with Hoechst 33342. As the cells die, the breakdown of the plasma membrane can be tracked by noting when the calcein stain disappears, which is followed almost immediately by breakdown of the nuclear membrane and PI positivity. Tis was not a 24-hour experiment, but a similar protocol could work for you. You need to ensure that the cells have a good control for phototoxicity and any other factors which can cause cell death. Chris Guerin chris.guerin@irc.vib-ugent.be


Along the same lines as Chris’s suggestion, DAPI also makes a


great viability marker. Benjamin Smith benjamin.smith@berkeley.edu Depending on what stage(s) of cell death or apoptosis you are


interested in tracking, there are a number of options to consider, each with their caveats beyond a quick thread here. Here’s a Methods paper (Measuring Apoptosis at the Single Cell Level, https://doi. org/10.1016%2Fj.ymeth.2007.11.007) I worked on with a few fantastic colleagues in Doug Green’s group more than a dozen years ago. Here’s another option from an Apoptosis Methods book that you might have access to (no free access that I can find) from that same time period: Live to Dead Cell Imaging (https://doi.org/10.1007/978-1-60327-017- 5_3). Sam Connell samuel.connell@gmail.com


Tank you all very much for your advice and for sharing your experience in this field. Best regards, Verona Villar vvillar@umh.es


Fluorescent Indicator for Membrane Damage Confocal Listserver Does anyone have suggestions for indicators of membrane damage


(possibly short-lived) in live cells? We are trying propidium iodide (PI), which is not supposed to enter intact live cells but may enter a damaged transiently damaged membrane and then light up in the nucleus. Te nice thing about PI is that it is not fluorescent until it associates with DNA or RNA. Are there other methods that people on the list can suggest? Tanks in advance. Aryeh Weiss aryeh@cc.huji.ac.il


68 doi:10.1017/S1551929521001048 FM dyes also work the same as PI and other DNA binding dyes


but leaves nucleic acids untouched. Teir fluorescence increases upon entering cells and binding intracellular membranes and the labeling intensity is proportional to the extent of damage. Here is our description of its use – https://doi.org/10.3791/51106. Jyoti Jaiswal jkjaiswal@cnmcresearch.org


I want to thank the many list members who replied both on


and off list. We received many suggestions, including using calcium indicators, a variety of suggestions for anionic dyes that do not enter intact cells, many DNA indicators in case we do not like PI, references on the use of PI for this purpose, and references from people who have done similar studies. So, we have a lot to chew on, and I can say that just about anything I might do with a scope has been done by someone (or many) on this list. Aryeh Weiss aryeh@cc.huji.ac.il


Nuclear Marking and Segmentation Confocal Listserver DAPI and similar fluorophores are regularly used to highlight nuclei,


but the staining is inhomogenous within nuclei and can vary substantially between nuclei in the same specimen. Tis makes segmentation for quantitative analysis difficult. So, any suggestions for a marker that produces a more uniform fluorescence within individual nuclei and less variation between nuclei, which would make image analysis simpler when we only want to know where the nuclei are. Jeremy Adler jeremy.adler@igp.uu.se


www.microscopy-today.com • 2021 September


I am not sure if this might help. Phosphatidylserine is not exactly


an indicator of plasma membrane damage, but with stress and loss of homeostasis it is externalized to the outer leaflet of the plasma membrane. Tis can be easily visualized with Annexin V conjugated to a fluorophore (there are many commercial sources of this reagent). Tis happens before propidium iodide can get into the cells and label DNA. Javier Diez Guerra fdiez@cbm.csic.es


Load the cells with calcein using Calcein-AM. Calcein leaks


out with membrane damage. Tis is the basis for commercial live- dead assays using calcein in combination with propidium iodide. John Lemaster lemaste@musc.edu


In flow cytometry, many dyes such as PI are used as viability dyes.


Examples include DAPI, 7-AAD, SYTOX, DRAQ7, etc. Tere are also fixable viability dyes from many manufacturers. And as mentioned by John, dyes such as Calcein-AM can be used as vital dyes to determine which cells are still metabolically active. One example of a publication mentioning this is: Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition) by Cossarizza et al., 2019 in Eur. J. Immunol. Chapter 3.4 “Dead cell exclusion, cell viability, and sample freezing” https://doi.org/10.1002/eji.201970107. Christian Kukat christian.kukat@age.mpg.de


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