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Apoptosis assay using fluorescent microscope rather than FACS


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#1 TTT

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Posted 31 May 2010 - 01:44 AM

I am going to use the ImageXpress micro instrument which is a automated fluorecent microcope to look at apoptosis/necrosis in cells and im going to use the Vybrant apoptosis assay kit 7 that uses Propidium iodide, Hoescht and YO-PRO-1 dyes. However the protocol i got from are for flow cytometry use, Im just not sure how i can adapt a protocol for flow cytometry to fluorescent microscope, i guess with ImageXpress, my cells are all attached to the plate whereas flow cytometry protocol, the cells are in solutions. So I have questions like do I remove the dyes before imaging, do i fix cells or not etc, has anyone done apoptosis study with fluorescent microcope rather than FACS and has a good protocol for me to adapt, thanks.

#2 Inmost sun

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Posted 01 June 2010 - 12:08 PM

I am going to use the ImageXpress micro instrument which is a automated fluorecent microcope to look at apoptosis/necrosis in cells and im going to use the Vybrant apoptosis assay kit 7 that uses Propidium iodide, Hoescht and YO-PRO-1 dyes. However the protocol i got from are for flow cytometry use, Im just not sure how i can adapt a protocol for flow cytometry to fluorescent microscope, i guess with ImageXpress, my cells are all attached to the plate whereas flow cytometry protocol, the cells are in solutions. So I have questions like do I remove the dyes before imaging, do i fix cells or not etc, has anyone done apoptosis study with fluorescent microcope rather than FACS and has a good protocol for me to adapt, thanks.


in deed, the same dys are also to use in fluorescent microscopy; Invitrogen offers various protocols for their dyes; you should do live cell imaging

#3 scamp

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Posted 07 June 2010 - 01:00 AM

I am going to use the ImageXpress micro instrument which is a automated fluorecent microcope to look at apoptosis/necrosis in cells and im going to use the Vybrant apoptosis assay kit 7 that uses Propidium iodide, Hoescht and YO-PRO-1 dyes. However the protocol i got from are for flow cytometry use, Im just not sure how i can adapt a protocol for flow cytometry to fluorescent microscope, i guess with ImageXpress, my cells are all attached to the plate whereas flow cytometry protocol, the cells are in solutions. So I have questions like do I remove the dyes before imaging, do i fix cells or not etc, has anyone done apoptosis study with fluorescent microcope rather than FACS and has a good protocol for me to adapt, thanks.



Have you tried the APOPercentage kit from Biocolor. It uses light microscopy and is quantifiable.

www.biocolor.co.uk

#4 moljul

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Posted 08 June 2010 - 06:13 AM

i agree with inmost sun!

use the same dye, also the FACS staining protocol you can perform for microscopical use. the stained cells can then be brought onto a slide using so called cytospin.

#5 scamp

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Posted 09 June 2010 - 01:38 AM

I am going to use the ImageXpress micro instrument which is a automated fluorecent microcope to look at apoptosis/necrosis in cells and im going to use the Vybrant apoptosis assay kit 7 that uses Propidium iodide, Hoescht and YO-PRO-1 dyes. However the protocol i got from are for flow cytometry use, Im just not sure how i can adapt a protocol for flow cytometry to fluorescent microscope, i guess with ImageXpress, my cells are all attached to the plate whereas flow cytometry protocol, the cells are in solutions. So I have questions like do I remove the dyes before imaging, do i fix cells or not etc, has anyone done apoptosis study with fluorescent microcope rather than FACS and has a good protocol for me to adapt, thanks.



#6 scamp

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Posted 09 June 2010 - 01:44 AM

I am going to use the ImageXpress micro instrument which is a automated fluorecent microcope to look at apoptosis/necrosis in cells and im going to use the Vybrant apoptosis assay kit 7 that uses Propidium iodide, Hoescht and YO-PRO-1 dyes. However the protocol i got from are for flow cytometry use, Im just not sure how i can adapt a protocol for flow cytometry to fluorescent microscope, i guess with ImageXpress, my cells are all attached to the plate whereas flow cytometry protocol, the cells are in solutions. So I have questions like do I remove the dyes before imaging, do i fix cells or not etc, has anyone done apoptosis study with fluorescent microcope rather than FACS and has a good protocol for me to adapt, thanks.



#7 scamp

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Posted 09 June 2010 - 01:45 AM

forgot to say
the apopercentage assay uses attached cells. You may be able to adapt the method.

APOPercentage manual

#8 TTT

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Posted 23 June 2010 - 03:17 AM

Thanks for the reply guys, I am going to use the Invitrogen dyes and adapt their flow cytometry protocol to fluorescent microscope protocol. I have decided to use propidium iodide, YO-PRO-1 and Hoest to discriminate apoptosis, necrosis and live cells, hope it works!




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