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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 <[log in to unmask]> wrote:
> 
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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 <[log in to unmask]>
> wrote:
> 
>> *****
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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" <[log in to unmask]> wrote:
>>> 
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>> posting.
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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
>>