Cell permeability assay *tech support response*

[Roy Edward-2]

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Dear Jens,

There have now been a number of papers citing the use of the far-red DNA
binding viability probe DRAQ7 for real-time and long-term monitoring of
cell health. This may be suitable for your application.  It¹s been shown
not to have inherent toxicity of its own in long-term exposure in a number
of recent publications. Importantly, a reporter of cell health like this,
that only marks the leaky (dead/damaged) cells, avoids unnecessary
perturbation that might come from a positive reporter of metabolic
competence or capacity.

These papers include:


Marciniak, A., Selck, C., Friedrich, B., & Speier, S. (2013). Mouse
pancreas tissue slice culture facilitates long-term studies of exocrine
and endocrine cell physiology in situ. PLoS One 8(11) e78706.
- 7-day culture of pancreas explant tissue



Ware, M. J., Godin, B., Singh, N., Majithia, R., Shamsudeen, S., Serda, R.
E., ... & Summers, H. D. (2014). Analysis of the influence of cell
heterogeneity on nanoparticle dose response. ACS nano, 8(7), 6693-6700.
- real-time nanoparticle toxicity monitoring; they used the DRAQ7 ³event²
to report each individual cell membrane failure in real-time, perhaps akin
to your assay need


Liang, J. R., Martinez, A., Lane, J. D., Mayor, U., Clague, M. J., & Urbé,
S. (2015). USP30 deubiquitylates mitochondrial Parkin substrates and
restricts apoptotic cell death. EMBO reports, e201439820.
- real time measurements (12, 72 h with time-lapse) of
mitochondrially-regulated apoptosis


Wang, J., Li, B., & Wu, M. X. (2015).
Effective and lesion-free cutaneous influenza vaccination. Proceedings of
the National Academy of Sciences, 112(16), 5005-5010.
- detecting subcutaneous extracellular dsDNA in better understanding its
adjuvant role in vaccination (fascinating stuff!)


All underpinned by a thorough exploration of the performance fundamentals
for such assays in:
Akagi,J., Kordon, M., Zhao, H., et al. (2013a). Real-time cell viability
assays using a new anthracycline derivative DRAQ7. Cytometry A 83, 227-234.

- including a highly sensitive assay for supravital DNA binding molecules
previously developed by Darzynkiewicz and colleagues showing it does not
induce DNA damage signaling. This further infers it does not equilibrate
inside intact cells given the nanomolar sensitivity of this assay.


This viability probe is DNA specific to give additional textural
information (that might reflect differences in cell death e.g.
catastrophic or programmed) which also benefits from being preferentially
red-excited, thereby limiting the risk of DNA damage associated with
shorter wavelengths (UV particularly) especially in time-lapse imaging. As
a far-red fluorescing viability probe has complete spectral separation
from the vast majority of other fluorescent reporters (e.g.
UV/vio-excited, fluorescein-, rhodamine-based).

I would recommend testing it at 0.1 - 1.0 uM for the concentration
required in your system.  This concentration is much lower than that
applied in the Akagi paper, which was more appropriate to endpoint
measurements by flow cytometry.  It should persist over many days without
experiencing chemical deterioration or photo-bleaching.

Full details are available at http://www.biostatus.com/DRAQ7 - technical
datasheets, reference lists, MSDS and app notes.

DRAQ7 (1 ml: Code DR71000) is available direct from BioStatus or from our
partner Cell Signaling Technology  (Product Number 7406S) in North America
(full distributor list:
http://www.biostatus.com/Customer-Support/Our-channel-Partners).


Otherwise, please contact me off-list and we can discuss details of
DRAQ7¹s possible incorporation into your assay.

Kind regards,
Roy

Roy Edward
E roy(at)biostatus(dot)com

BioStatus Limited
56a Charnwood Road, Shepshed, Leics. LE12 9NP U.K.
T [phone]
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>------------------------------
>Date:    Thu, 6 Aug 2015 16:42:29 -0400
>From:    Jens-Bernhard Bosse <[hidden email]>
>Subject: Cell permeability assay
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>Dear List,
>I would like to image the spread of a virus through a monolayer. The =
>virus is fluorescently tagged such that infected cells can be easily =
>followed.
>At some point late in infection, cells will loose their membrane =
>integrity and I would like to record that timepoint.
>Now comes my question: Does anybody know a cell permeability marker that =
>can be left in the culture medium for days and does not affect the =
>cells? Trypan Blue?
>Thanks!
>Jens