Robust high-throughput kinetic analysis of apoptosis with real-time high-content live-cell imaging.

Dual-reporter method for kinetic analysis of apoptotic progression A common requirement of cell death research is the measure of early (stable exposure of PS on the extracellular leaflet of the plasma membrane) and late apoptotic events (loss of plasma membrane integrity), which in flow cytometry-based protocols is commonly accomplished by dual staining with Annexin V and propidium iodide (for example, AV + PI − staining indicates early events while double positivity indicates late events). 13 However, PI is incompatible with our high-content long-duration imaging assay and is toxic with prolonged exposure (data not shown). Therefore, we screened several recently developed viability dyes (DRAQ7 and YOYO3) for compatibility within our assay and no toxicity. MEFs treated with inducers of apoptosis (CHX, or the pan-kinase inhibitor staurosporine, STS) were incubated with either DRAQ7 or YOYO3. In response to the apoptotic stimuli, more cells stained faster with YOYO3 than DRAQ7 ( Figures 2a and c ), and YOYO3 efficiently labelled cells at lower concentrations ( Figure 2d ). Finally, a comparison of DRAQ7 and YOYO3 exposure from 8 to 24 h revealed a twofold increase in apoptosis as measured by Annexin V-staining ( Figure 2e ). Collectively, these data suggest YOYO3 is an appropriate, non-toxic viability dye suitable for prolonged incubation in this new method. Next, we integrated YOYO3 and Annexin V staining in high-content cell imaging for real-time kinetic detection of apoptosis. MEFs were treated with apoptotic stimuli in the presence of Annexin V-488 and YOYO3 and imaged every 2 h for 24 h. As shown in Figures 3a–c , Annexin V more rapidly detects apoptotic cells compared to YOYO3; similarly, YOYO3 fails to detect an apoptotic population before 8 h ( Figures 3c and d ). However, time points representing the onset of late apoptotic events demonstrate equivalent labelling by both Annexin V and YOYO3, further emphasizing the importance of collecting kinetic data ( Figure 3d ). The differences between Annexin V and YOYO3 labelling were verified by inspecting photographs captured by the high-content imager at the indicated times ( Figure 3e ). Interestingly, cells treated as above but subjected to preparation for flow cytometry demonstrated no differential labelling between Annexin V and YOYO3 ( Figure 3f ). We suspect this is due to physical stress applied during sample preparation resulting in the incorporation of YOYO3 prior to the cellular dismantling of the plasma membrane. The majority of apoptosis detection assays require caspase activation; as such, caspase-cleavable peptide reporters (for example, the conserved caspase-3/-7 cleavage site derived from PARP, D-E-V-D) are commonly used in high-throughput assays. Next we compared the sensitivity of Annexin V to a DEVD reporter by treating MEFs with apoptotic stimuli in the presence of both detection reagents. When directly compared, Annexin V staining occurred more rapidly and on more cells than the DEVD peptide ( Figure 3g ). Furthermore, Annexin V demonstrated robust photostability while both the DEVD peptide and YOYO3 photobleached during data acquisition over 48 h ( Figure 3h ). Collectively, these data suggest that Annexin V is a superior labelling reagent for kinetic analysis. Additionally, while YOYO3 does photobleach it appropriately couples with Annexin V staining in high-content cell imaging to reveal early and late apoptotic events in one assay for both single-cell ( Figure 3e ) and population studies ( Figure 3c ). Up to this point, we examined common inducers of apoptosis to test the validity of this methodology. Next we investigated additional treatments that engage distinct pro-apoptotic signalling mechanisms. Apoptosis occurs through two pathways: the extrinsic pathway, which is activated by extracellular ligands (for example, Tumour Necrosis Factor Alpha, TNF α ) that bind to death receptors; and the intrinsic pathway, which is engaged by cellular stress (for example, terminal Unfolded Protein Response activated by Tunicamycin, Tn, or Thapsigargin, Tg). 14 , 15 , 16 , 17 Activation of the extrinsic pathway via treatment with TNFα or death-receptor ligand TRAIL resulted in rapid Annexin V labelling and cell death when co-treated with CHX, which prevents translation of pro-survival proteins ( Figure 4a , black line; Supplementary Figure S2A ). As a control to ensure Annexin V-positivity was the result of apoptosis, MEFs were co-treated with modulators of the apoptotic pathway, which caused the expected acceleration (ABT-737, an inhibitor of anti-apoptotic proteins) or inhibition of apoptosis (zVAD-fmk, a pan-caspase inhibitor), respectively ( Figure 4a , red and brown lines). 18 , 19 Within these assays, YOYO3-positivity always followed Annexin V-positivity by a few hours and demonstrated a similar pattern of apoptotic kinetics ( Figure 4b , Supplementary Figure S2B ), and comparable staining efficiency in cells achieving late-stages of apoptosis ( Figure 4c ). Consistent with our previous results, similarly-treated cells analysed by flow cytometry failed to reveal differential staining between Annexin V and YOYO3, and we suspect the physical stress during processing promotes YOYO3 incorporation ( Figure 4d ). As a note, STS-treated cells demonstrated variable labelling by either Annexin V or YOYO3 ( Figure 4e ) and therefore use of TNFα and CHX or CHX and ABT are more suitable positive controls to measure maximal and accelerated cell death. Similar to the results obtained with death receptor ligands ( Figures 4a–e ), MEFs displayed similar kinetics of apoptosis when treated with inducers of UPR, ABT-737 and zVAD-fmk ( Figures 4f and j ). 20 In these assays, however, the delay between Annexin V- and YOYO3-positivity was markedly pronounced compared to inducers of the extrinsic pathway ( Figures 4h and i ). Furthermore, different cell types demonstrate signature kinetic patterns between early and late apoptotic events (data not shown). As high-throughput cytotoxic assays are frequently used for drug or RNAi screens, which engage the intrinsic pathway of apoptosis, these data suggest that the use of cell viability dyes in high-content cell imaging is imperfect due to an inaccurate and markedly delayed labelling of apoptotic populations. In contrast, the described Annexin V-labelling is highly sensitive, responsive and accurate for measuring the onset of apoptosis.

Materials and methods

Reagents and equipment

Cell culture reagents and chemicals were from Sigma Aldrich (St. Louis, MO, USA) or Thermo Fisher Scientific (Waltham, MA, USA) unless otherwise stated. Drugs and biologics as follows: ABT-737 (Abbott Pharmaceuticals, Lake Bluff, IL, USA); Tn, Tg, CHX and STS (Sigma); DRAQ7 (Abcam, Cambridge, UK); YOYO3, CellTrace CFSE, DyeCycle Green and CellEvent Caspase-3/-7 Green Detection Reagent (Thermo Fisher Scientific); mTNFα and mTRAIL (Peprotech, Rocky Hill, NJ, USA); zVAD-fmk (ApexBio, Houston, TX, USA). Recombinant Annexin V was purified, labelled with FITC, DyLight 633 NHS ester (Thermo Fisher Scientific), or AlexaFluor 594 NHS ester (Thermo Fisher Scientific), and re-purified using Dye Removal Columns (Thermo Fisher Scientific) following conjugation. Purity, stability and fluorescence of labelled recombinant proteins were analysed by SDS-PAGE.

Fluorescent imaging of labelled recombinant Annexin

V was accomplished using an MP ChemiDoc Imager equipped with LED excitation cubes (Bio-Rad, Hercules, CA, USA).

Tissue culture and cell preparation

Experiments were performed with SV40-transformed MEFs, U266, HeLa or primary MEFs where labelled. Cells were cultured in DMEM containing 10% FBS, 2 mM L-glutamine and antibiotics; U266 cells were cultured in RPMI 1640 containing 10% heat-inactivated FBS, and antibiotics. For kinetic studies, cells were seeded at approximately 3–5 × 10 3 cells/well in a 96-well format or 8–10 × 10 3 cells/well in a 48-well format for 24 h. Prior to live-cell imaging, media was replaced with phenol-red free complete DMEM containing: indicated treatments, DRAQ7 or YOYO3, and fluorescent Annexin V variants. For experiments utilizing DyeCycle (1 μg/ml in PBS) or CellTrace (1 μM in PBS), cells were incubated at 37 °C for 30 min before washing and replaced with the phenol-red free complete DMEM. Plates were pre-warmed prior to data acquisition to avoid condensation and expansion of the plate, which hinders autofocus. Experiments utilizing RPMI media, which contains 0.4 mM Ca 2+ , required CaCl 2 supplementation for robust Annexin V labelling (data not shown).

Flow cytometry

Cells were plated in 48-well plates at 1.5 × 10 4 , cultured overnight, and treated as described. At the time of collection, supernatants were collected and adherent cells were trypsinized for 5–10 min. Cells were pipetted up and down in trypsin to aid in detachment before combining with corresponding supernatant. Cells were pelleted via centrifugation at 1100 × g for 15 min and decanted. Unlike traditional Annexin V-binding protocols, cells were not resuspended in ABB but instead in complete DMEM to avoid cytotoxic effects of ABB. Staining was performed in DMEM containing Annexin V-488 (1 μg/ml) as well as YOYO3 (200 nM) for dual detection by flow cytometry.

Data acquisition and analysis

Kinetic experiments were performed with the IncuCyte ZOOM (Essen Bioscience, Ann Arbor, MI, USA), but other high-content live-cell fluorescent imagers are capable of replicating these data. Experiments were conducted for 20–48 h with data collection every 2–4 h to avoid photobleaching of fluorescent reporters. Using the × 10 objective, four planes of view were collected per well for experiments using a 48-well plate; a single plane of view was collected for 96-well plate assays. Phase contrast, green channel (Ex: 440/80 nm; Em: 504/44 nm), and red channel (Ex: 565/05 nm; Em: 625/05 nm) were collected for all experiments with spectral unmixing set as 5% of red removed from green. Processing definitions are described below ( Table 1 ). Data were always exported as events per well. Conversion of data to apoptotic percentages used the following algorithm (mathematically depicted below): raw percentages for each time point were calculated as Annexin V-positive events divided by the CellTrace value (Equation 1 ); an adjustment factor was calculated by dividing the difference between minimum and maximum percentages of all data points by 100 (Equation 2 ); each set of triplicate raw percentages was averaged and divided by the adjustment factor to obtain an adjusted percent from 0 to 100 inclusive (Equation 3 ). where: R =raw percentage , F =adjustment factor, P =adjusted percentage, i =arbitrary well, j =arbitrary time, w o =first well, w f =last well, t o =initial time, t f =final time, A =Annexin V value, =average CellTrace value, i/i’ / i’’ =triplicate well set, [ w o ,w f ]=inclusive range of well values, [ t o , t f ]=inclusive range of time data for wells.

Supplementary Material Supplementary Figure 1 Click here for additional data file. Supplementary Figure 2 Click here for additional data file. Supplementary Figure 3 Click here for additional data file. Supplementary Figure 4 Click here for additional data file.