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.
-TTT-
TTT on May 31 2010, 09:44 AM said:
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
-Inmost sun-
TTT on May 31 2010, 01:44 AM said:
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
-scamp-
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.
-moljul-
TTT on May 31 2010, 02:44 AM said:
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.
-scamp-
scamp on Jun 9 2010, 02:38 AM said:
TTT on May 31 2010, 02:44 AM said:
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.
-scamp-
forgot to say
the apopercentage assay uses attached cells. You may be able to adapt the method.
APOPercentage manual
-scamp-
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!
-TTT-
