Use of Draq 7 with Nile Red

[Shane van Breda]

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Dear group,

I would like to try the following and I am hoping someone can give me insight.

I will be incubating M. tuberculosis in presence of an antibiotic for 24 hours. I will
use it at the determined MIC and 2 x MIC.

I will remove the cells, centrifuge to obtain a pellet, rinse and resuspend it. I will
stain with Draq 7 (3uM) and allow for the reaction to take place at room
temperature for 30 min.

Once stained with Draq 7, I will centrifuge and concentrate the pellet (100ul) and
spot it onto Poly - L - Lysine slides (20 ul). I will fix and sterilise the slide using
formaldehyde fumes (25 % v/v) over night (previously established protocol).

Next, I would like to rinse the slide, allow it to dry, then stain the slide with Nile
Red, followed by rinsing.

I want to view Nile red at Ex ~ 488nm, Em ~ 525 and Draq 7 at Ex ~ 633 or 647 ,
Em ~ 695 LP.

My concern is that there will be an overlap with Draq 7, but I am not 100 % sure?

Thanks in advance

[Craig Brideau]

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Nile Red has a long spectral tail and can exhibit spectral shifts under
various conditions. Have you considered Prodan or similar as an
alternative?

Craig Brideau
On Sep 8, 2014 8:04 AM, "Shane van Breda" <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear group,
>
> I would like to try the following and I am hoping someone can give me
> insight.
>
> I will be incubating M. tuberculosis in presence of an antibiotic for 24
> hours. I will
> use it at the determined MIC and 2 x MIC.
>
> I will remove the cells, centrifuge to obtain a pellet, rinse and
> resuspend it. I will
> stain with Draq 7 (3uM) and allow for the reaction to take place at room
> temperature for 30 min.
>
> Once stained with Draq 7, I will centrifuge and concentrate the pellet
> (100ul) and
> spot it onto Poly - L - Lysine slides (20 ul). I will fix and sterilise
> the slide using
> formaldehyde fumes (25 % v/v) over night (previously established protocol).
>
> Next, I would like to rinse the slide, allow it to dry, then stain the
> slide with Nile
> Red, followed by rinsing.
>
> I want to view Nile red at Ex ~ 488nm, Em ~ 525 and Draq 7 at Ex ~ 633 or
> 647 ,
> Em ~ 695 LP.
>
> My concern is that there will be an overlap with Draq 7, but I am not 100
> % sure?
>
> Thanks in advance
>

[jerie]

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Shane,

because of the characteristic double peak emission of Draq7, I would
presume it is ez to separate from Nile Red using spectral unmixing. It is
not clear to me though why you would choose this combination of
fluorophores?
The peak emission of Nile red depends on the ligand but will certainly be
longer than 525nm, according to -for example- life tech spectral viewer.

no commercial interests in either of the products

kind regards, jens

Visiting Scientist @ Center for Technological Development in Health (CDTS),
Oswaldo Cruz Foundation (Fiocruz), Ministry of Health, Rio de Janeiro,
Brazil.
http://br.linkedin.com/pub/jens-rietdorf/6/4a3/189/
Skype jens.rietdorf


On Mon, Sep 8, 2014 at 1:32 PM, Craig Brideau <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Nile Red has a long spectral tail and can exhibit spectral shifts under
> various conditions. Have you considered Prodan or similar as an
> alternative?
>
> Craig Brideau
> On Sep 8, 2014 8:04 AM, "Shane van Breda" <[hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Dear group,
> >
> > I would like to try the following and I am hoping someone can give me
> > insight.
> >
> > I will be incubating M. tuberculosis in presence of an antibiotic for 24
> > hours. I will
> > use it at the determined MIC and 2 x MIC.
> >
> > I will remove the cells, centrifuge to obtain a pellet, rinse and
> > resuspend it. I will
> > stain with Draq 7 (3uM) and allow for the reaction to take place at room
> > temperature for 30 min.
> >
> > Once stained with Draq 7, I will centrifuge and concentrate the pellet
> > (100ul) and
> > spot it onto Poly - L - Lysine slides (20 ul). I will fix and sterilise
> > the slide using
> > formaldehyde fumes (25 % v/v) over night (previously established
> protocol).
> >
> > Next, I would like to rinse the slide, allow it to dry, then stain the
> > slide with Nile
> > Red, followed by rinsing.
> >
> > I want to view Nile red at Ex ~ 488nm, Em ~ 525 and Draq 7 at Ex ~ 633 or
> > 647 ,
> > Em ~ 695 LP.
> >
> > My concern is that there will be an overlap with Draq 7, but I am not 100
> > % sure?
> >
> > Thanks in advance
> >
>

[Shane van Breda]

*****
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*****

I would like to use the combination because my antibiotic makes cell walls permeable.

I am interested in seeing a correlation between cell wall permeability or death with Draq 7 and correlating that to the inclusion or loss of lipid inclusions.

Nile red methods for viewing lipid inclusions in MTB is well established.

I'm beginning to think it will be safer to use them seperately.

Thanks,

Shane

> On 08 Sep 2014, at 8:55 PM, jens rietdorf <[hidden email]> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Shane,
>
> because of the characteristic double peak emission of Draq7, I would
> presume it is ez to separate from Nile Red using spectral unmixing. It is
> not clear to me though why you would choose this combination of
> fluorophores?
> The peak emission of Nile red depends on the ligand but will certainly be
> longer than 525nm, according to -for example- life tech spectral viewer.
>
> no commercial interests in either of the products
>
> kind regards, jens
>
> Visiting Scientist @ Center for Technological Development in Health (CDTS),
> Oswaldo Cruz Foundation (Fiocruz), Ministry of Health, Rio de Janeiro,
> Brazil.
> http://br.linkedin.com/pub/jens-rietdorf/6/4a3/189/
> Skype jens.rietdorf
>
>
> On Mon, Sep 8, 2014 at 1:32 PM, Craig Brideau <[hidden email]>
> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> Nile Red has a long spectral tail and can exhibit spectral shifts under
>> various conditions. Have you considered Prodan or similar as an
>> alternative?
>>
>> Craig Brideau
>>> On Sep 8, 2014 8:04 AM, "Shane van Breda" <[hidden email]> wrote:
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> Post images on http://www.imgur.com and include the link in your
>> posting.
>>> *****
>>>
>>> Dear group,
>>>
>>> I would like to try the following and I am hoping someone can give me
>>> insight.
>>>
>>> I will be incubating M. tuberculosis in presence of an antibiotic for 24
>>> hours. I will
>>> use it at the determined MIC and 2 x MIC.
>>>
>>> I will remove the cells, centrifuge to obtain a pellet, rinse and
>>> resuspend it. I will
>>> stain with Draq 7 (3uM) and allow for the reaction to take place at room
>>> temperature for 30 min.
>>>
>>> Once stained with Draq 7, I will centrifuge and concentrate the pellet
>>> (100ul) and
>>> spot it onto Poly - L - Lysine slides (20 ul). I will fix and sterilise
>>> the slide using
>>> formaldehyde fumes (25 % v/v) over night (previously established
>> protocol).
>>>
>>> Next, I would like to rinse the slide, allow it to dry, then stain the
>>> slide with Nile
>>> Red, followed by rinsing.
>>>
>>> I want to view Nile red at Ex ~ 488nm, Em ~ 525 and Draq 7 at Ex ~ 633 or
>>> 647 ,
>>> Em ~ 695 LP.
>>>
>>> My concern is that there will be an overlap with Draq 7, but I am not 100
>>> % sure?
>>>
>>> Thanks in advance
>>