Re: Use of Draq 7 with Nile Red **Tech support response**

[Roy Edward-2]

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Hi Shane,
It should be relatively easy to image the two signals in a time resolved
manner for each chromophore since Nile Red will not be excited by the red
source required for DRAQ7.

Though, depending on the filter cube used, there should not be bleed
through from blue excited Nile Red (NR, which has very broad emission)
into the DRAQ7 far-red channel especially if a Cy7 emission filter is
available (780/60).  It might need to be borne in mind whether the
detector is NIR sensitive.

Also, there may be different patterning between the NR and DRAQ7 signals
in a membrane-compromised cell which could be informative.

I hope that helps.
Kind regards,
Roy Edward

E roy(at)biostatus(dot)com

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Date:    Mon, 8 Sep 2014 09:03:04 -0500
From:    Shane van Breda <[hidden email]>
Subject: Use of Draq 7 with Nile Red

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Dear group,

I would like to try the following and I am hoping someone can give me ins=
ight.

I will be incubating M. tuberculosis in presence of an antibiotic for 24 =
hours. I will=20
use it at the determined MIC and 2 x MIC.

I will remove the cells, centrifuge to obtain a pellet, rinse and resuspe=
nd it. I will=20
stain with Draq 7 (3uM) and allow for the reaction to take place at
room=20=

temperature for 30 min.

Once stained with Draq 7, I will centrifuge and concentrate the pellet (1=
00ul) and=20
spot it onto Poly - L - Lysine slides (20 ul). I will fix and sterilise t=
he slide using=20
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 sl=
ide with Nile=20
Red, followed by rinsing.

I want to view Nile red at Ex ~ 488nm, Em ~ 525 and Draq 7 at Ex ~ 633 or=
647 ,=20
Em ~ 695 LP.

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

Thanks in advance