发信人: John (Peter), 信区: Biology
标 题: Re: Questions about JC-1 staining
发信站: Unknown Space - 未名空间 (Fri Oct 8 14:26:59 2004) WWW-POST

The emmission wavelength of JC-1 is 590, it is orange (for most people).
For ideal flow cytometry, you have to change your filter system to get
optimal result. For confocal microscopy, try to put in approiate filter
(usually it's hard), or you might need to do some real good compensation
(sometime
difficult, too). So, the third choise for confocal will be monitoring only one
channel (decrease of orange or increase of green)

If the nuclear is stained, that means your JC-1 concentration in the cell
is too high. Decrease the concentration of the dye or give a couple of more
times (say 2-3) washs will help.

All these mithondrial potential dyes are really "potential" dyes. They will
also measure membrane potential. Therefore, JC-1 will be concentrated in
mitochondria, then released to the cytosol, and eventually release out of
the cell. To detect repoliarization, you really need to supply the dye
in the culture buffer. But because of the toxicity of JC-1 for long term
culture and their "overloading" problem, very few people used them. The right
choise will be TMRM (or TMEM or something.) All these dyes are available in
molecular probes (www.probes.com)


【 在 bewithu (x-fire in the sea) 的大作中提到: 】
:
:
: I used JC-1 to stain macrophage. In live cells, the emission light is not
red,
:
: but orange. Why? What is more, the live cells show green using another
filter
:
: (I do not know the wavelengh, sorry). Many papers show brighly red light
after
:
:
: JC-1 staining in live cells, and only the cytoplams be stained. I am
confused
:
: why my cells are orange and the light distribute in the nucleus also.
:
: My second question is that if I pre-stain live cells with JC-1 and induce mt

: depolarization, will the red light disappear and green light form?
: And if cells with depolarized mt are stained with JC-1, and then add sth to

: make the mt polarized again, will the the monomer JC-1 go into mt?
:
: Thank u very much
:
:


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