Biotek Cytation™5 + DRAQ7™ for real-time cell death

A new application note authored by Brad Larson, Principal Scientist at Biotek Instruments, Inc. showcases their integrated BioSpa platform, which includes the Cytation™5, and the combination of three fluorescent probes to unpick and quantify mechanisms of cell death. 

The methodologies described should see wide applicability for the investigation of unwanted drug toxicity, cancer chemotherapeutic screening, personalised medicine, cell-based therapy and immunotherapy.  Far-red DRAQ7™ was selected as the reporter of cells with compromised membranes - signifying necrosis, mechanically-induced cell damage or a later feature of apoptosis.  The three reagents used were all chosen for their compatibility with real-time/time-lapse imaging

Automated, Multi-Parameter, Kinetic Methods to Quantify Cell Death - Application Note
Brad Larson, Principal Scientist, BioTek Instruments, Inc., Winooski, VT USA. March, 2018

An integrated, automated cell incubation and imaging workflow was used to demonstrate a multi-parametric and kinetic methodology to assess cell death: the BioSpa Live Cell Imaging System consists of the Cytation™5 Cell Imaging Multi-Mode Reader and BioSpa 8 Automated Incubator (Biotek Instruments, Inc.).

In the test system, fibrosarcoma HT1080 cells were treated with a 7-point, 4-fold dilution series of Camptothecin in the presence of TMRE (Abcam), pSIVA-IANDB (Abcam), and DRAQ7™ to detect mitochondrial membrane potential, externally-exposed phosphatidylserine and compromised cell membranes / necrosis respectively.  Wells were imaged at 2-hourly intervals for 48 hours for the three chromophore signals and for brightfield to assess cell numbers.

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Real-time cell health monitoring in 2D/3D with DRAQ7 - latest papers

DRAQ7™ has become the choice for image-based real-time / time-lapse cell health monitoring.   The following papers add to the evidence base on the product's broad applicability and high performance:

Spheroid / 3D Culture Cell Health Analysis:

Dunlop, E. A., Johnson, C. E., Wiltshire, M., Errington, R. J., & Tee, A. R. (2017). Targeting protein homeostasis with nelfinavir/salinomycin dual therapy effectively induces death of mTORC1 hyperactive cells. Oncotarget, 8(30), 48711.

In cancer therapy research, scientists at Cardiff University have used DRAQ7™ to show the significance of discriminating between cytostatic and cytotoxic anti-tumour treatments.  Experiments showed these can be uncovered independent of 3-D spheroid diameter where after removal of cytostatic treatment spheroid growth may recover, indicating an "escape" from the intended outcome of the therapy.

In these experiments spheroids were exposed to DRAQ7™ for cell health monitoring for 36h, and with spheroid diameter was the only parameter required to observe post-cytostatic recovery or cytotoxic collapse.

Yu, K. K. H., Taylor, J. T., Pathmanaban, O. N., Youshani, A. S., Beyit, D., Dutko-Gwozdz, J., ... & Bigger, B. W. (2018). High content screening of patient-derived cell lines highlights the potential of non-standard chemotherapeutic agents for the treatment of glioblastoma. PloS one, 13(3), e0193694.

In the search for improved prognoses for glioblastoma patients clinical and academic researchers in Manchester, UK have compared 2D and 3D (neurosphere) assay formats for physiological relevance. Patient-derived cell lines were challenged with a panel of 83 drugs FDA-approved for a variety of other cancers to determine if there was useful cytotoxicity that might point to benefit in a personalised medicine approach to treatment.

The high content screen of cell/neurosphere exposure to a 5-log dilution series of each of the drugs for 96 h, and a final incubation with Hoechst 33342 (to count cells/neurospheres) and DRAQ7™ to determine cell death).

Sigmundsson, K., Ojala, J. R., Öhman, M. K., Österholm, A. M., Moreno-Moral, A., Domogatskaya, A., ... & George, B. (2018). Culturing functional pancreatic islets on α5-laminins and curative transplantation to diabetic mice. Matrix Biology, 70, 5.

This work from Duke-NUS Medical School, Singapore and Karolinska Institute, Stockholm showed the potential for a sustained reversal of Type 1 Diabetes by transplantation of islets allowed to spread onto basement membrane protein α5-laminin coated PDMS membrane.

These islets showed excellent viability and functional capacity over many days.  In contrast, islets maintained in suspension culture rapidly developed large hypoxic (MAR+), necrotic cores DRAQ7™+) which were shown to be expelled, as shown in striking video and static images.

Lee, S. W. L., Adriani, G., Ceccarello, E., Pavesi, A., Tan, A. T., Bertoletti, A., ... & Wong, S. C. (2018). characterizing the role of Monocytes in T cell cancer immunotherapy Using a 3D Microfluidic Model. Frontiers in immunology, 9, 416.

To investigate the inhibitory role of monocytes in T cell cancer immunotherapy researchers at A-STAR, Singapore and MIT, USA developed a 3D intrahepatic tumor microenvironment microfluidic model.  Importantly they showed that in the equivalent 2D system monocytes did not inhibit T-cell killing of target hepatocytes, suggesting that the 3D model was a superior and more physiologically relevant system.

HepG2 hepatocytes were tagged with GFP, T-cells labelled with Cell Tracker Violet and resulting dead cells monitored by DRAQ7™ in the culture medium, with the three fluorescence signals measured by confocal z-stack fluorescence imaging at 0h and 24h.  Dead HepG2 cells were defined as GFP-dim, DRAQ7™+ objects. Monocytes were not labelled.

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2-D cell Health Assays

Szemes, M., Greenhough, A., Melegh, Z., Malik, S., Yuksel, A., Catchpoole, D., ... & Malik, K. (2018). Wnt signalling drives context-dependent differentiation or proliferation in neuroblastoma. bioRxiv, 236745.

In a study into Wnt and its target genes in neuroblastoma (NB), researchers at the University of Bristol have described transcriptionally distinct features that determine a preference for differentiation or proliferation in response to Wnt signaling.

To study the proliferative response, NB cell lines were monitored in the IncuCyte ZOOM (Essen Biosciences) for 24 hours, in the presence of 1.5 µM DRAQ7™, and following Wnt stimulus, imaged every 2 hours for progress to confluence and for a far-red signal denoting each individual dead cell.

Deo, P., Chow, S. H., Hay, I. D., Kleifeld, O., Costin, A., Elgass, K. D., ... & Lithgow, T. (2018). Outer membrane vesicles from Neisseria gonorrhoeae target PorB to mitochondria and induce apoptosis. PLoS pathogens, 14(3), e1006945.

To study the modulation of the host innate immune system by N. gonorrhoeae, researchers at Monash University, Australia allowed macrophages to ingest outer-membrane vesicles (OMV) rich in a Porin PorB.

In time-lapse imaging experiments they demonstrated that Porin B targets mitochondria and leads to caspase-dependent (apoptotic) cell death, in a time-dependent manner. This was determined by a combination of the observed sequence of collapsing TMRM signal (mitochondrial membrane potential loss), transient Cell Event Caspase 3/7 signal (Cytochrome C release and consequent caspase activation) and finally a persistent far-red viability probe DRAQ7™ signal in the nucleus of each cell over a 48 hour time course, monitored every 30 minutes.

Automated, Multi-Parameter, Kinetic Methods to Quantify Cell Death - Application Note
Brad Larson, Principal Scientist, BioTek Instruments, Inc., Winooski, VT USA. March, 2018

An integrated, automated cell incubation and imaging workflow was used to demonstrate a multi-parametric and kinetic methodology to assess cell death. The BioSpa Live Cell Imaging System consists of the Cytation™5 Cell Imaging Multi-Mode Reader and BioSpa 8 Automated Incubator (Biotek Instruments, Inc.).

In the test system, fibrosarcoma HT1080 cells were treated with a 7-point, 4-fold dilution series of Camptothecin in the presence of TMRE (Abcam), pSIVA-IANDB (Abcam), and DRAQ7™ to detect mitochondrial membrane potential, externally-exposed phosphatidylserine and compromised cell membranes / necrosis respectively.  Wells were imaged at 2-hourly intervals for 48 hours for the three chromophore signals and for brightfield to assess cell numbers.


Previous methodologies have positively marked cells based on positive metabolic competence.  Conversely, DRAQ7™ labels only membrane-compromised (damaged, dying, dead) cells, its red excitation minimises risk of DNA damage when capturing multiple time-lapse images whilst reliably monitoring viability in real-time, cell-by-cell.

DRAQ7™ offers new dimensions and opportunities for performance of high value phenotypic and in vitro toxicity cell-based assays in drug discovery and development, that can be applied across different platforms including flow cytometry, imaging flow cytometry, fluorescence microscopy and high content imaging platforms.

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DRAQ7 - more sorted than even we thought!

Researchers' confidence in DRAQ7™ as the viability probe for single cell sorting shown in a growing citation list.

Since we published our white paper on single cell sorting (available here) in Spring 2018 there have been several more articles that we believe merit attention..

1. Sorting for molecular analysis  e.g. RNA-seq
2. Sorting for downstream cell culture and assay

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1. Sorting for molecular analysis
Jackson, L, et al. "Incomplete inhibition of HIV infection results in more HIV infected lymph node cells by reducing cell death." eLife 7 (2018): e30134.
This work highlights a delicate balance that the infecting HIV virus aims to achieve by infecting as many new cells as possible while killing the orginating cell of de novo virions. It points to a potential risk of using therapeutics to treat partially resistant viral strains, that exacerbated infection and effects of HIV patients.  To enumerate HIV DNA copies per cell, cells were single-cell sorted a BD Biosciences FACSAria III sorter.  GFP-positive, DRAQ7-negative RevCEM clones were sorted 1 day post-infection into 96 well plates for downstream PCR analysis.

Govindan, S, Oberst, P., & Jabaudon, D. "In vivo pulse-labeling of isochronic cohorts of cells in the central nervous system using FlashTag." bioRxiv (2018): 286831.
A new protocol to label, track and isolate newborn cells in the CNS in vivo. CFSE labelling (termed FlashTag) of progenitors in the embryonic CNS persists post-natally such that cells can be analyzed ex vivo or in fixed tissue, for electrophysiology, or FACS-sorted for cell culture or (single-cell) RNA-sequencing.  When cells were isolated by FACS sorting, DRAQ7 was used to exclude dead and damaged cells from collection, downstream use and analysis.  Sorting was performed on the Beckman Coulter Moflo Astrios cell sorter.

Humbert, M, et al. "Intratumoral CpG-B Promotes Antitumoral Neutrophil, cDC, and T-cell Cooperation without Reprograming Tolerogenic pDC." Cancer research 78.12 (2018): 3280-3292.
In an effort to affect immune tolerance towards tumours i.t. CpG-B (a TLR-9 agonist) was found to aid the tumour recruitment of neutrophils as tumour-associated conventional dendritic cells (TA-cDC) but did not re-programme resident plasmacytoid dendritic cells (TA-pDC), which were refractory to the treatment.   To understand the gene expression profiles of the TA-pDC and TA-cDC cells were sorted on the basis of phenotype and exclusion of dead cells (DRAQ7+).  Gene expression confirmed that the TA-cDC have upregulated gene expression associated with antitumour T-cell immunity, in contrast to the down-modulation in TA-pDC.  Sorting was performed on the BioRad S3 Sorter.

Klingler, E, et al. "A translaminar genetic logic for the circuit identity of intracortically-projecting neurons." bioRxiv (2018): 290395.
This work shows intracortically-projecting neurons (ICPN) have transcriptional identities primarily in accord with input-output relationships rather than time of origin or laminar position.  It appears there are conserved circuit-related transcriptional programs across cortical layers and that these may be maintained circuit features through development and evolution.  For single sorting for downstream RNA-sequencing, fresh tissue was micro-dissected from regions of interest, digested and sieved.  Singlet cells, positive for Hoechst and negative for DRAQ7, were sorted on the Beckman Coulter Moflo Astrios cell sorter for cDNA synthesis.

2. Sorting for downstream cell culture and assay
Handgraaf, S, et al. "17-β Estradiol regulates proglucagon-derived peptide secretion in mouse and human α-and L cells." JCI insight 3.7 (2018).
This article investigates the potentially beneficial role of the estrogenic pathway and, specifically of ERβ agonists to prevent type 2 diabetes.  Human islets were dissociated with accutase and α and β cells sorted according to staining or not with an antibody against α-cell surface marker HPa1, excluding doublets and dead cells (DRAQ7+).  Cells were cultured independently, treated with test compounds and controls and then co-cultured and assayed for GLP-1 and Insulin release.  Sorting was performed on the BioRad S3 Sorter.

DRAQ7's unique constellation of properties make it the choice when live-sorting cells for downstream analysis, to exclude dead and damaged cells without risk of contamination of the intact cells, of interference of polymerase function or of cell culture potential. 

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The CryoChem Method - optimal morphology meets correlative imaging (CLEM)

The CryoChem Method (CCM) is a new hybrid approach to the processing of cryofixed cells and tissues that enables preservation of ultrastructural morphology while being compatible with genetic EM tags, fluorescent proteins and counterstaining, and onwards to SBEM and CLEM.

The research, led by Professor Mark Ellisman, UCSD, benchmarks this new approach in a variety of samples to test its broad applicability.  The work, firstly shows that, following rehydration, a genetically-encoded EM tag (peroxidase APEX2) is able to accumulate electron-dense DAB.  Next, the quantitative measurement of two structurally different fluorescent proteins GFP and tdTomato is demonstrated.

To explore the opportunities presented further in correlated light and electron microscopy (CLEM), a mouse brain slice expressing tdTomato in specific neurons is processed by CCM and the ability to counterstain the nuclei tested, using the far-red DNA probe DRAQ5™.  This is established successfully at a typical concentration (5 µM), on ice for 60 minutes.  Thereafter the tissue slice is subjected to en bloc staining with heavy metals and embedded in readiness for serial block-face electron microscopy (SBEM).  The block is then analyzed by micro-CT to correlate this X-ray volume to the DRAQ5™-stained nuclei as fiducial markers of the confocal imaging data to determine a region of interest for SBEM imaging of tdTomato neurons.

It is also possible to correlate confocal and SBEM data using the bright chromatin labelling by DRAQ5™ to the matching finer SBEM features and the tdTomato fluorescence to similarly delineate cell bodies and the neuronal processes.

The authors suggest that this method should also be compatible with the earlier ChromEMT method (see separate blog) which uses DRAQ5™ to photo-catalyze the accumulation of DAB to chromatin and thereby enabling OsO4 labelling for EM tomography (EMT).

This new work further extends the breadth of use for DRAQ5™ as a nuclear counterstain.

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Reference:
Tsang, T. K., Bushong, E. A., Boassa, D., Hu, J., Romoli, B., Phan, S., ... & Ellisman, M. H. (2018). High-quality ultrastructural preservation using cryofixation for 3D electron microscopy of genetically labeled tissues. eLife. 2018; 7: e35524

Read more articles on DRAQ5™ in exciting new EM techniques: 

• Simplified CLEM - from in vivo imaging to FIB/SEM
• ChromEMT for EM-Tomography of chromatin - direct labelling of chromatin

Drosha-dependent DNA damage response

Fascinating new research led by scientists at the MRC Toxicology Unit (Leicester, UK) explores a key and non-canonical role for Drosha in the DNA Damage Response (DDR). 

In wide-ranging studies Lu et al. show the dependence of DDR early in the response, upstream of the decision to repair double-strand breaks (DSB) using homologous recombination (HR) or non-homologous end-joining (NHEJ), which appear to predominantly occur during G2/M or G1/O phases of the cell cycle respectively, and perhaps reflecting the demand for error-free nature of HR during de novo DNA synthesis or in regions with increased transcriptional activity.

In one cell-based assay, Drosha (and Dicer) were subjected to siRNA knockdown in U2OS cells, which were then subjected to radiomimetic DNA damaging agent bleomycin.  Cells were then probed with Annexin V(-FITC) and DRAQ7™ to determine the proportion of cells in late apoptosis by flow cytometry (BD Biosciences FACSCanto II), which was markedly greater in the cells with knockdown of either Drosha or Dicer.

In such cell-based assays far-red fluorescing viability probe DRAQ7™ shows excellent separation between intact cells and those with compromised membranes.  It has no spectral overlap with Annexin V-FITC, used in this work.  This pairing can be further combined with a mitochondrial membrane potential probe TMRM which can together aid interrogation of the cell death mechanism involved.

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Reference:
Lu, W. T., Hawley, B. R., Skalka, G. L., Baldock, R. A., Smith, E. M., Bader, A. S., ... & Bushell, M. (2018). Drosha drives the formation of DNA: RNA hybrids around DNA break sites to facilitate DNA repair. Nature communications, 9(1), 532.

Vanishing GFP? Here's the answer..

Around the world, scientists just like you have pondered how to preserve the signal from their GFP expressing cells after having watched weeks of work disappear before their very eyes..

Fixation procedures can allow leakage out of the GFP-tagged protein or be directly detrimental to the fragile amino acid cage that enables GFP's remarkable properties, properties that literally started a cell biology revolution and rightly recognised by a Nobel Prize!

Q. How can we reliably measure GFP expression in cells by flow cytometry or by imaging?

A. Analyse LIVE, intact cells that we've stained with DRAQ5™ or CyTRAK Orange™.

These two cell-permeant DNA binding probes are spectrally compatible with GFP and allow you to counterstain your cells, alive and intact, without any unnecessary processing with fixative or permeabilization.  If necessary, you can use a much gentler fixation (e.g. less formaldehyde) and that remains compatible with these probes.  (High content screening labs even combine fixative and DRAQ5 to have only one pipetting step during the last steps prior to imaging).

Counterstaining is rapid (15 min.) and no washing is required.  Just tamp away any excess liquid from your cells on a slide, apply any required mountant and coverslip prior to imaging.

For flow cytometry and imaging flow cytometry the nuclear staining will be the last step, with no need for washing, proceeding directly to the analyzer.

Which one should you choose?  This spectrum might help.

Searchlight™is provided courtesy of Semrock/IDEX Health & Science, LLC

Of course, your equipment options may determine this for you, but for either of these counterstains you won't be waiting to book time on a UV-equipped 'scope or cytometer! To make it easier, here's a quick guide to each of the products for microscopy, flow cytometry and imaging flow cytometry respectively:

DRAQ5™ has peak absorbance at 599 and 646 nm so any of the longer wavelength excitation sources will suit for microscopy - 594, 635, 647 nm - alongside 488 nm excitation for your GFP measurement.

DRAQ5™'s emission is detected above 670 nm so your microscope or high content imager needs to have a far-red channel.  This is commonly a 675LP (long-pass), often called the "Cy5" channel but it could be any broad bandpass (BP) filter sitting in the 670-750 nm range.

This means that your precious GFP and DRAQ5™ counterstain signals can be captured simultaneously (ideal for HCS) from two independent excitation sources.

Remarkably, in modern flow cytometers DRAQ5™ can be efficiently excited by blue through red lasers and is detected in a channel similar to those described above for microscopy.

On the Imagestream®x (Merck) imaging flow cytometer DRAQ5™ is detected in channel 11 (Camera 2) or channel 5 (Camera 1).

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However, if your microscope only has blue laser excitation or cannot detect far-red emission signals or you've got another red/far-red fluorophore in your flow analysis that cannot be compensated for..

CyTRAK Orange™ has peak absorbance at 515 nm so is best excited by 488 nm or 532 nm sources. CyTRAK Orange™'s peak emission is centred on 615 nm so a filter/channel previously designated for propidium iodide is ideal.

Here then you can excite GFP and CYTRAK Orange™ simultaneously off the same excitation source for ideal pixel-pixel registration and without needing a UV-source or far-red detection - meaning a simple wide-field fluorescence microscope should be sufficient.

For flow cytometry, CyTRAK Orange™ can be excited by the blue laser line (488 nm) and detected in the channel otherwise specified for propidium iodide.

On the Imagestream®x imaging flow cytometer we recommend detecting CyTRAK Orange™ in channel 4 (Camera 1).

AND, as added bonus..

You can use these cell permeant probes for your fixed cell preps and FFPE tissue sections as well, already cited in thousands of publications.

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Using the ImagestreamX? Here's a new counterstaining option

Are you using the Imagestream®x (ISX) imaging flow cytometer from Merck?
If you are, then you will probably have used DRAQ5™ at some point in your research work!

There are more than 300 publications citing use of DRAQ5™ on Imagestream 100 and x platforms.

Why..?

• DRAQ5™ gives clean nuclear labeling - enabling compartment segmentation, morphometrics, study of translocations, cell-cell interactions, micronuclei detection, and many more as well as an obvious trigger for nucleated cells and setting best focusing.
• DRAQ5™ can be used on intact (or fixed) cells - it rapidly labels nuclear DNA without permeabilisation or fixation.  This is fantastic if you're trying to measure GFP, taking away the worry that fixation kills GFP fluorescence or leakage out of the cell. (See a recent example.)
• DRAQ5™ is far-red fluorescing - it can be used in combination with a wide range of chromophores and viability and functional dyes.

The typical concentration range is 1-5 µM, with exceptions for special applications such as micronucleus detection requiring higher amounts to achieve the stoichiometry needed.

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Q. BUT what if a cell feature needs a chromophore in the channel you detect DRAQ5™?

A. Switch to CyTRAK Orange™!

• CyTRAK Orange™ is a closely related chemical relative of DRAQ5™. 
• CyTRAK Orange™ is cell permeant and labels the nucleus (like DRAQ5™) but it is optimally excited at 515 nm and can be detected in ISX's channel 4 (595-660 nm).  
• CyTRAK Orange™ is NOT excited by the red laser line.
• CyTRAK Orange™ doesn't have spectral overlap with GFP. 

The spectra below show where CyTRAK Orange™ is situated compared to selected relevant chromophores, including eGFP.

We suggest a working concentration of 3-10 µM for most applications.

Note: CyTRAK Orange™ can be combined with DRAQ7™ for a convenient live/dead combination as used by Pieper et al. Cytometry Part A 2016.

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Searchlight™is provided courtesy of Semrock/IDEX Health & Science, LLC
Imagestream is a registered trademark of Merck KGaA