Monday, 12 November 2018

New DRAQ5 citations in drug discovery

The far-red cell permeant DNA dye DRAQ5™ has been widely used as a nuclear counterstain in high content screening (HCS) applications for more than a decade.  It is compatible with all common HCS imaging platforms and flow cytometers.

Why screeners choose DRAQ5:
  • It gives good nuclear segmentation but also gives a useful secondary cytoplasmic signal permitting demarcation of the cytoplasmic envelope, in live- or fixed-cell endpoints
  • Far-red - it is spectrally separated from GFP and most vis-range chromophores
  • It is not excited by the UV/violet laser thereby avoiding compound interference
  • Stable on the automation deck allowing longer unattended operation 
  • 1 ml of DRAQ5 is sufficient for 100,000 wells (1536-mtp) or 33,000 wells (384-mtp)
Here is an extensive review of the most recent papers in high content screening / drug discovery citing the use of DRAQ5™ with a snapshot of the topic and technique relevant to the use of DRAQ5.  The papers are organised according to the imaging platform used and are from 2017 and 2018. 

Analysis Platform: Perkin Elmer Opera™ / Opera Phenix™

Spheroid imaging (Perkin Elmer Opera Phenix™ (and Operetta™ CLS))
Foitzik, Angelika, et al. "More Content from High-Content Screening: Analyzing Spheroids in 3D." Poster Presentation. SLAS Europe 2018, Brussels.
HeLa cell spheroids - untreated, and cleared with ScaleS4 and ScaleA2. The cleared spheroids were counterstained with 4 µM DRAQ5 for 2h (fixed, if not cleared) and imaged.

Leishmania infection of macrophages imaging assay (Imaging with Opera™ QEHS)
Hefnawy, Aya, et al. "Importance of secondary screening with clinical isolates for anti-leishmania drug discovery." Scientific reports 8.1 (2018): 11765.
THP-1 cells were infected with counterstained amastigotes and then treated with compounds. Finally cells were prepared with a combined fix & stain solution (8% formaldehyde & 5 µM DRAQ5) prior to imaging.  Nuclei were localised, cells segmented and amastigotes enumerated using the DRAQ5 staining pattern.

Anti-apoptotic Bax phoshorylation in cancer therapy resistance (Opera Phenix)
Kale, Justin, et al. "Phosphorylation switches Bax from promoting to inhibiting apoptosis thereby increasing drug resistance." EMBO reports 19.9 (2018): e45235.
For live cell endpoint imaging, WT and transfected BMK cells were variously treated and then stained with Annexin V-FITC, TMRE and DRAQ5 (5 µM) for 30 minutes prior to imaging.

Anti-parasitic active compound screening (Opera)
Cogo, Juliana, et al. "Quinoxaline derivatives as potential antitrypanosomal and antileishmanial agents." Bioorganic & medicinal chemistry 26.14 (2018): 4065-4072.
H9c2 cells infected with trypomastigotes and treated with a compound library
Counterstained with a fix & stain solution (8% formaldehyde & 4 µM DRAQ5) prior to imaging.
Assay scored on i) host cell nuclei (compound toxicity), ii) amastigotes per host cell (level of infection) and iii) percentage host cells infected (secondary infection measure).

Unpicking myocardium-GPCR signaling (Opera Phenix)
Martin, Ryan D., et al. "Receptor-and cellular compartment-specific activation of the cAMP/PKA pathway by α1-adrenergic and ETA endothelin receptors." Cellular signalling 44 (2018): 43-50.
HEK 293 cells transfected with BRET reporters of cAMP, specific for either cytoplasm or nucleus, were treated with agonists, fixed with 4% formaldehyde (10 min.) and counterstained with DRAQ5, (10 min., conc. not stated).  

Druggable target for SLE.. (Opera)
Brightbill, Hans D., et al. "NF-κB inducing kinase is a therapeutic target for systemic lupus erythematosus." Nature communications 9.1 (2018): 179.
HeLa cells were screened with compounds for nuclear translocation of either RelA (with TNF stimulation) or p52. Cells were fixed (4% formaldehyde) and permeabilised (PBS/0.1% Triton X-100), probed with the appropriate primary antibody and then stained with Alexa Fluor 488 secondary antibody and DRAQ5 (incubation details not disclosed).

NCE leads for ebola virus (Opera)
Schafer, Adam, et al. "Repurposing potential of 1st generation H1-specific antihistamines as anti-filovirus therapeutics." Antiviral research 157 (2018): 47-56.
HFF-1 or HeLa cells were exposed to ebola virus and then treated with a compound library.  Cells were fixed with formalin, probed with ebola glycoprotein- specific primary antibody and subsequently with DyLight 488-tagged secondary antibody and finally DRAQ5 (incubation details not disclosed).  DRAQ5 staining was used to segment the nucleus and cytoplasm and infection level scored for each compound's anti-viral activity.

Requirements for optimal DSB repair in the DNA damage response (Opera)
Baranes-Bachar, Keren, et al. "The ubiquitin e3/e4 ligase ube4a adjusts protein ubiquitylation and accumulation at sites of DNA damage, facilitating double-strand break repair." Molecular cell 69.5 (2018): 866-878.
Two siRNA screens targeting E1, E2, E3- Ubiquitin ligases and players in the Ubiquitin-proteasome system were performed with outputs for KAP-1 phosphorylation and 53BP1 foci.  Reverse transfection was performed on f-89 hTERT cells, then treated with radiomimetic or inhibitors of ATM or DNA-PK.  Following the treatment incubation period cells were fixed (4% formaldehyde) and permeabilised (0.5% Triton X-100) and probed with primary antibody against pKAP-1 or 53BP1 and fluorochrome-tagged secondary antibody before nuclear counterstaining with DRAQ5 (2.5 µM, 20 min. at room temp.).

Initial cell state influences gene expression leading to heterogeneity (Opera Phenix)
Fritzsch, Christoph, et al. "Estrogen‐dependent control and cell‐to‐cell variability of transcriptional bursting." Molecular systems biology 14.2 (2018): e7678.
Estrogen response was measured on complex GFP transfections of MCF-7 cells cells with various small molecule inhibitors. Cells were fixed (4% formaldehyde, 10 min., 4 degC) and then counterstained with DRAQ5 (2.5 µM, 30 min.).

Anti-flavivirus natural compound with identified host target (Opera)
Estoppey, David, et al. "The natural product cavinafungin selectively interferes with Zika and dengue virus replication by inhibition of the host signal peptidase." Cell reports 19.3 (2017): 451-460.
In high content screens dengue virus infected A549 cells were exposed to canivafungin to establish IC50 & CC50 then HUH7 (Cas9 stably-transfected) cells were exposed to CRISPR/Cas9 genetic knockout and then dengue virus infected.  In both screens, cells were fixed and probed with DyLight 488-tagged primary antibody against flavivirus envelope protein (4G2).  Thereafter, cells were counterstained with DRAQ5.

Host cell immune response exploration (Opera)
Polajnar, Mira, et al. "Expanding the host cell ubiquitylation machinery targeting cytosolic Salmonella." EMBO reports 18.9 (2017): 1572-1585.
A siRNA knockdown for RBR E3 ligases was performed on HeLa cells, which were then exposed to S. typhimurium (GFP+, under cytosolic control). After a period of infection the cells were fixed (4% formaldehyde) and permeabilised (0.1% Triton X-100) and in one emulation cells were then probed with anti-ubiquitin antibody and finally counterstained with DRAQ5 (1 µM, 10 min., ambient) for imaging.


Analysis Platform: Perkin Elmer Operetta™

Sequential staining of organelles in live cells (Operetta)
Alamudi, Samira Husen, et al. "A palette of background-free tame fluorescent probes for intracellular multi-color labelling in live cells." Chemical science 9.8 (2018): 2376-2383.
U2OS cells were exposed to alkyne:azide paired fluorescent reporter and organelle probe or alkyne-containing fluorescent reporter after azide-containing sugars incorporated into the cell surface glycan.  After washout of excess reagent live cells were stained with DRAQ5 (1 µM, 30 min. 37 degC) and imaged.

New targets in cancer cell metabolism (Operetta)
Thongon, Natthakan, et al. "Cancer cell metabolic plasticity allows resistance to NAMPT inhibition but invariably induces dependence on LDHA." Cancer & metabolism 6.1 (2018): 1.
To assess the effects on mitochondrial health and numbers, parental and FK866-resistant CCRF-CEM and MDA-MB231 cells were treated with FK866.  After treatment cells were incubated for 30 min. with DRAQ5 and MitoTracker™ Orange (100 nM; Invitrogen). Intensity of mitochondrial staining and their numbers/cell were assessed by imaging while the addition of FCCP and mock allowed reporting of mitochondrial function loss.

Anchored ErbB2 antibodies - equivalent performance at lower titres (Operetta)
Milazzo, Ferdinando Maria, et al. "AvidinOX-anchored biotinylated trastuzumab and pertuzumab induce down-modulation of ErbB2 and tumor cell death at concentrations order of magnitude lower than not-anchored antibodies." Oncotarget 8.14 (2017): 22590.
To assess the effect of AvidinOX-anchored antiErbB2 therapeutic antibodies, SKBR3, BT474 and MCF7 cells were probed for altered expression of a wide range of anti-apoptotic and pro-apoptotic markers. Cells were fixed (4% formaldehyde) and permeabilized (PBS 0.2%Tween-20) and labelled indirectly with FITC-tagged secondary antibodies or directly conjugated FITC-, Alexa Fluor 488-, PE- and Alexa Fluor 555-tagged primary antibodies.  β-Galactosidase activity was measured with a fluorescent probe C₁₂FDG (Molecular Probes).  In each case thereafter, cells were counterstained with DRAQ5 and plates read by high content imaging.

Zika virus infection screening method (Operetta)
Koishi, Andrea Cristine, et al. "Development and evaluation of a novel high-throughput image-based fluorescent neutralization test for detection of Zika virus infection." PLoS neglected tropical diseases 12.3 (2018): e0006342.
Huh 7.5 cells were exposed to virus and inactivated patient sera to screen for presence of anti-viral neutralising antibodies.  After incubation, cells were fixed with cold methanol:acetone and probed with monoclonal 4G2 (anti-ZIKV envelope protein) and indirectly detected by AlexaFluor 488-tagged secondary antibody and finally counterstained with DRAQ5 (5 µM).

Natural compound discovery in new places (Operetta)
Demers, Danielle, et al. "Exploitation of Mangrove Endophytic Fungi for Infectious Disease Drug Discovery." Marine drugs 16.10 (2018): 376.
Thomas, Santana AL, et al. "Keikipukalides, Furanocembrane Diterpenes from the Antarctic Deep Sea Octocoral Plumarella delicatissima." Journal of natural products 81.1 (2017): 117-123.
In screens for anti-parasitic effects, macrophages (J774.A1) were infected with L. donovani amastigotes.  Following incubation in the presence of positive and negative controls and fungal extracts cells were fixed (2% formaldehyde; 15 min.) and counterstained with DRAQ5 (5 µM; 5 min.) to disclose the macrophage nuclei, cytoplasm and score the amastigotes in the cytoplasm.

SAR for a new anti-Chagas' Disease compound series (Operetta)
Palace-Berl, Fanny, et al. "Investigating the structure-activity relationships of N’-[(5-nitrofuran-2-yl) methylene] substituted hydrazides against Trypanosoma cruzi to design novel active compounds." European journal of medicinal chemistry 144 (2018): 29-40.
To test biological effect of compounds, U2OS cells were infected with trypomastigotes and on day 3 cells were treated with the compound series in a dilution scheme.  Following 72 h incubation cells were fixed (4% formaldehyde) and counterstained with DRAQ5 (5 µM) to show host cell number (including unwanted cytotoxicity cf. DMSO control), infection rate and parasites per cell.  

New anti-trypanosome flavonoid scaffolds (Operetta)
Di Pisa, Flavio, et al. "Chroman-4-One Derivatives Targeting Pteridine Reductase 1 and Showing Anti-Parasitic Activity." Molecules 22.3 (2017): 426.
To assess anti-Leishmania infantum activity of compounds, THP-1 cells were infected with promastigotes for 24h and then exposed to compounds and controls and incubated for a further 48h.  Cells were then fixed (4% formaldehyde) and counterstained with DRAQ5 to enumerate host cell number, infection ratio and parasites per cell.

Refining an imaging assay for anti-Chagas' Disease screens (Operetta)
Yang, Gyongseon, et al. "Evaluation of parameters impacting drug susceptibility in intracellular Trypanosoma cruzi assay protocols." SLAS Discovery 22.2 (2017): 125-134.
An improved modification of an image-based assay showed excellent correlation with the whole-well readout colorimetric assay.  In short, U2OS cells were seeded in plate wells for 24h and then infected with trypomastigotes for 48 h. Thereafter compounds were added and assessed after 48 h or 72 h for effects.  Prior to imaging, cells were fixed 4% formaldehyde), washed to remove external parasites and counterstained with DRAQ5 to reveal U2OS host cell nuclear and parasite DNA, detected at 633nm excitation and 690 nm emission.  The separate seeding of cells and parasites and 72 h compound exposure delivered the excellent inter-assay correlation.


Analysis Platform: Thermo Arrayscan™

High throughput toxicity assay - live/dead analysis of colonospheres (Arrayscan VTi)
Trumpi, Kari, et al. "Paired image-and FACS-based toxicity assays for high content screening of spheroid-type tumor cell cultures." FEBS open bio 5 (2015): 85-90.
Patient-derived colonospheres were treated with anti-cancer compounds.  Colonospheres were then treated with verapamil and then Calcein Green AM (4 µM) and DRAQ5 (2 µM) added, incubated for 10 min. at 37 degC.  Colonspheres were spun down prior to imaging.  For each DRAQ5 DNA-positive event (live or dead cell) the CalceinGreen AM signal was acquired to give an output for the cellularity and live:dead ratio for individual colonospheres.

Cigarette smoke exposure - effect on monocyte-endothelial cell interactions (Arrayscan)
Poussin, Carine, et al. "Systems Biology Reveals Cigarette Smoke-Induced Concentration-Dependent Direct and Indirect Mechanisms That Promote Monocyte–Endothelial Cell Adhesion." Toxicological Sciences 147.2 (2015): 370-385.
Hoechst 33342-labeled human coronary human coronary artery endothelial cells (HCAEC) were cultured with MM6-conditioned medium or controls, exposed to cigarette smoke extract and controls.  Thereafter, DRAQ5-labeled (at 5 µM) MM6 cells (a monocyte-like cell line) were overlaid on the HCAECs and incubated to permit adhesion (37 degC, 45 min.), washed 3 times then fixed (4% formaldehyde).  The adhesion of DRAQ5+ MM6 cells was scored a percentage of the HCAECs.


Analysis Platform: Molecular Devices ImageXpress

Investigating pericyte function by culture medium used (ImageXpress Micro XLS)
Rustenhoven, Justin, et al. "Modelling physiological and pathological conditions to study pericyte biology in brain function and dysfunction." BMC neuroscience 19.1 (2018): 6.
Testing for different responses to DMEM/F12 and a defined pericyte medium. In one assay for phagocytosis, fluorescent bead engulfment was measured per cell with DRAQ5 as counterstain, incubated for 30 minutes (post-fixation with 4% formaldehyde).

Antibody-drug conjugate therapy in breast cancer (ImageXpress Micro)
Kelly, Marcus P., et al. "Preclinical activity of the novel anti-Prolactin Receptor (PRLR) antibody-drug conjugate REGN2878-DM1 in PRLR positive breast cancers." Molecular cancer therapeutics (2017): molcanther-0839.
To test for internalization of PRLR antibody REGN2878, Alexa Fluor 488-tagged REGN2878 was incubated with MCF-7/PRLR and T47D on ice and at 37 degC.  After 1 h cells were washed and counterstained with DRAQ5 and imaged for the presence of intracellular antibody fluorescence.

Optimizing gain-of-function reverse transfections (ImageXpress Ultra)
Kim, Hi Chul, et al. "Optimization of Cell-Based cDNA Microarray Conditions for Gene Functional Studies in HEK293 Cells." SLAS DISCOVERY 22.8 (2017): 1053-1059.
HEK 293 cells were reverse transfected with cDNAs for gain-of-function.  To image the performance of this, GFP transfected cells were fixed (4% formaldehyde, 10 min.) washed and counterstained with DRAQ5 (2.5 µM).

Peptomers as novel antibiotics (ImageXpress Ultra)
Lam, Hanh, et al. "Synthetic cyclic peptomers as type III secretion system inhibitors." Antimicrobial agents and chemotherapy (2017): AAC-00060.
CHO-K1 cells were infected with Yersinia sp. transduced with a beta-lactamase reporter for translocation of T3SS effector proteins in the presence of peptomers or vehicle (DMSO).  After 1 h to permit infection, cells were incubated with lactamase substrate (30 min., RT) and then fixed (4% formaldehyde, 20 min.) and then counterstained with DRAQ5.  Positive translocation was shown by blue fluorescence cf. green fluorescence.


Analysis Platform: Merck ImageStream™and FlowSight

Automating micronucleus detection by imaging flow cytometry
Rodrigues, M. A., et al. "Multi‐parameter dose estimations in radiation biodosimetry using the automated cytokinesis‐block micronucleus assay with imaging flow cytometry." Cytometry Part A 85.10 (2014): 883-893. (Imagestream)
Verma, Jatin R., et al. "Investigating FlowSight® imaging flow cytometry as a platform to assess chemically induced micronuclei using human lymphoblastoid cells in vitro." Mutagenesis 33.4 (2018): 283-289. (FlowSight)
Whole peripheral blood was subjected to radiation exposure (to simulate a mass radiological event e.g. Fukushima, 2011) and lymphoblastoid cell lines MCL-5, TK-6 were treated with known DNA damaging chemicals, with both regimens known to generate micronuclei as a measure of genotoxicity.  Following each regimen samples were treated identically in both articles:  cells were pelleted and resuspended in BD FACSlyse solution (12 min., RT) for RBC lysis/fixation or fixation respectively, washed and then stained with DRAQ5 (50 µM; 5 or 30 min.; RT) and analysed directly without washing.  DRAQ5 was excited at 488 nm (FlowSight) and 658 nm (ImageStream).  Single and multi-nucleated cell events with and without micronuclei could be identified and scored automatically.

Dimethyl Fumarate (DMF) reduces BBB endothelial transmigration in MS (Imagestream)
Breuer, Johanna, et al. "Dual action by fumaric acid esters synergistically reduces adhesion to human endothelium." Multiple Sclerosis Journal (2017): 1352458517735189.
Nucelar translocation of NFκB was determined in treated CD4+ T cells by permeabilization for indirect labelling of the antigen with primary polyclonal (anti-p65 subunit) and secondary Alexa Fluor 488.  Cells were then counterstained with DRAQ5 (1 µM) and flow-imaged using the nuclear mask to determine the degree of translocation under different treatments and vehicle only.

PARP-1 inhibition increases radiation sensitivity in BRCA1 cancers (ImageStreamX MarkII)
Bourton, Emma C., et al. "The PARP-1 inhibitor Olaparib suppresses BRCA1 protein levels, increases apoptosis and causes radiation hypersensitivity in BRCA1+/-lymphoblastoid cells." Journal of Cancer 8.19 (2017): 4048.
To monitor the suppression of BRCA-1 protein expression and apoptosis on radiation exposure with and without Olaparib, five B-lymphoblastoid cell lines (normal: GM00893, GM05423; BRCA1 gene mutations: GM13705, GM14090, GM16105) were analysed by indirect immunofluorescence staining with anti-BRCA1 monoclonal antibody and Alexa Fluor 488 secondary antibody.  For this, cells were fixed in methanol:acetone and stored at -20 degC for later batch analysis and then re-hydrated (PBS) and permeabilized (0.5% Triton X-100).  Following antibody staining, cells were resuspended in in Accumax (Merck) and DRAQ5 added for counterstaining (50 µM) and flow-imaging to assess BRCA1 foci (Alexa Fluor 488 - Channel 2) and apoptosis (as determined by nuclear fragmentation and DNA condensation via DRAQ5 - Channel 5)).

Targeted cancer therapy - the value of protein abundance (ImagestreamX)
Adlung, Lorenz, et al. "Protein abundance of AKT and ERK pathway components governs cell type‐specific regulation of proliferation." Molecular systems biology 13.1 (2017): 904.
BaF3‐EpoR (lymphoid) and 32D‐EpoR (myeloid) cell lines and isolated murine and human CFU‐E progenitors were treated with Epo +/- inhibitors. They were subsequently analysed for volumetric changes by imaging flow cytometry by staining the cell nucleus with DRAQ5 and cytoplasm with calcein.

PDGFRβ pathway - a new target for Retinoblastoma therapies (FlowSight)
Goldsmith, Zachary K., et al. "Targeting the Platelet-Derived Growth Factor-beta Stimulatory Circuitry to Control Retinoblastoma Seeds." Investigative ophthalmology & visual science 59.11 (2018): 4486-4495.
To measure NFκB translocation in Y79 cells were treated with Gleevec/imatinib or control (and further with PDGFRB siRNA knockdown to confirm pathway) and harvested. They were fixed (2% formaldehyde) and permeabilized  (0.01% Triton X-100) for indirect immunofluorescence staining (AlexaFluor 488) of p65 subunit of NFκB and nuclei counterstained with DRAQ5 (50 µM) for subsequent flow-imaging analysis.


Further reading:
Smith, Paul J., Roy Edward, and Rachel J. Errington. "Recent advances in flow cytometry: Platforms, tools, and challenges for data analysis." Flow Cytometry in Drug Discovery and Development (2010): 23-54.
Edward, Roy. "Applications and Caveats on the Utilization of DNA-Specific Probes in Cell-Based Assays." High Content Screening. Humana Press, New York, NY, 2018. 3-19.

Technical datasheet and other key documents can be found on the DRAQ5 product page.

Read independent product reviews on DRAQ5, moderated by SelectScience.