hoechst 33342 nuclear dna stain  (Thermo Fisher)

Journal: Nature protocols

Article Title: Use of human induced pluripotent stem cell-derived cardiomyocytes to assess drug cardiotoxicity

doi: 10.1038/s41596-018-0076-8

Figure Lengend Snippet: Initially, hiPSCs will exhibit a tightly packed colony morphology and will express stem cell markers such as NANOG (green) and Tra-1–81 (red). Nuclear DNA stain DAPI is also shown (blue). This differentiation approach requires small molecule-mediated modulation of the Wnt signaling pathway by GSK3β inhibitor CHIR99021 and Wnt inhibitor Wnt-C59 to drive hiPSCs toward a cardiac mesoderm and cardiomyocyte fate. Beating hiPSC-CMs are typically visible by day 8 of differentiation. After cardiomyocyte differentiation and metabolic selection are complete, hiPSC-CMs will be pure and will express cardiomyocytespecific markers such as cardiac troponin T (cTnT; green) and alpha-actinin (red). Nuclear DNA stain DAPI is also shown (blue). At this point, they can be replated into high-throughput plates for downstream drug treatment and analysis. Medium changes (Δ) should be conducted on hiPSC-CMs every 2 d. NANOG antibody (Abcam, cat. no. AB21624) and Tra-1–81 antibody (EMD Millipore, cat. no. MAB4381) were utilized at 1:200 dilutions (~5 μg/mL) for hiPSC immunofluorescence staining. Cardiac troponin T antibody (Abcam, cat. no. AB45932) and alpha-actinin antibody (Sigma, cat. no. A7811) were utilized at 1:200 dilutions (~5 μ/mL) for hiPSC-CM immunofluorescence staining. For further details on how immunofluorescence and hiPSC-CM differentiation were conducted, refer to our previous study, Sharma et al.10. The CDM3 added to RPMI1640 in this figure refers to the CDM3 supplement.

Article Snippet: For these stains, 0.5 μM calcein-AM (Thermo Fisher Scientific, cat. no. C3100MP) or 5 μg/mL WGA (Thermo Fisher Scientific, cat. no. {"type":"entrez-nucleotide","attrs":{"text":"W11261","term_id":"1285566","term_text":"W11261"}} W11261 ) was combined with 2 μg/mL nuclear DNA stain (blue) Hoechst 33342 (Thermo Fisher Scientific, cat. no. H3570) in CDM3 basal medium pre-warmed to 37 °C.

Techniques: Staining, Selection, High Throughput Screening Assay, Immunofluorescence

Journal: Nature protocols

Article Title: Use of human induced pluripotent stem cell-derived cardiomyocytes to assess drug cardiotoxicity

doi: 10.1038/s41596-018-0076-8

Figure Lengend Snippet: a, A typical 384-well plate containing purified hiPSC-CMs. Insets illustrate optimal hiPSC-CM plating density via a cell body stain (calcein-AM; green) and a cell boundary stain (WGA, green). For these stains, 0.5 μM calcein-AM (Thermo Fisher Scientific, cat. no. C3100MP) or 5 μg/mL WGA (Thermo Fisher Scientific, cat. no. W11261) was combined with 2 μg/mL nuclear DNA stain (blue) Hoechst 33342 (Thermo Fisher Scientific, cat. no. H3570) in CDM3 basal medium pre-warmed to 37 °C. To optimize survival, hiPSC-CMs should be plated at high confluency as shown. Scale bars, 100 μm (left); 20 μm (right). b, Diagram illustrating one format for drug treatment in a 384-well plate, which allows for up to 24 drugs to be tested in duplicate, at eight half-log concentrations from 0 to 100 μM. c, Drug treatment workflow for cytotoxicity assessment in hiPSC-CMs in a 384-well plate. After exposure to drugs, quantitative viability assays such as PrestoBlue can quantify drug-induced alterations in hiPSC-CM viability. From these data, drug dose-response curves can be generated in a statistics program such as GraphPad Prism. In these example graphs, each colored line signifies an hiPSC-CM cell line derived from a unique individual. N = 5 independent biological replicate cytotoxicity assays were conducted for each cell line and drug, with error bars representing standard error. LD50 data can be extrapolated from these dose-response curves. The calcein-AM stain (green) indicates viable cells but is not required for cytotoxicity assessment. The Hoechst stain (blue) indicates DNA in cell nuclei. d, Drug treatment workflow for contractility assessment in hiPSC-CMs in a 384-well plate. After exposure to drugs, cells are stained with WGA (green) and Hoechst nuclear DNA stain (blue) so that they can be tracked using a platform such as the Vala Sciences KIC, optimized for high-throughput contractility imaging. Next, hiPSC-CM displacement analysis can be conducted to generate graphs illustrating hiPSC-CM movement in response to drug treatment over time. From this analysis, various contractility parameters can be extracted, including time (T) parameters such as time to peak (Tpeak) and TCT. Parameters also include displacement (D) parameters such as peak displacement (Dpeak). Heatmaps can be generated for each parameter, detailing how parameters change in response to drug concentration. Red coloration indicates a decrease in contraction rate, whereas green indicates an increase in contraction rate. Another parameter obtained from contractility analysis is cessation of beating, indicating the drug concentration that causes a majority of replicate wells to cease beating. WGA, wheat germ agglutinin. Portions of this figure were adapted with permission from Sharma et al.10, American Association for the Advancement of Science.

Article Snippet: For these stains, 0.5 μM calcein-AM (Thermo Fisher Scientific, cat. no. C3100MP) or 5 μg/mL WGA (Thermo Fisher Scientific, cat. no. {"type":"entrez-nucleotide","attrs":{"text":"W11261","term_id":"1285566","term_text":"W11261"}} W11261 ) was combined with 2 μg/mL nuclear DNA stain (blue) Hoechst 33342 (Thermo Fisher Scientific, cat. no. H3570) in CDM3 basal medium pre-warmed to 37 °C.

Techniques: Purification, Staining, Generated, Derivative Assay, High Throughput Screening Assay, Imaging, Concentration Assay