Cells grown on 25 mm glass coverslips were treated 24 hours with PBS, 1 mg/ml AGE-HSA or 1 mg/ml CML-HSA. The cells were treated with PBS or 200 nM AVP five min prior to fixation with 4.0% paraformaldehyde (G6403, Sigma) in PBS for 10 min and at room temperature. The fixed cells were washed with PBS for five min and permeabilized with 0.1% Triton X-100 (807426; MP Biomedicals) in PBS for one min. The cells were then incubated with the blocking solution Image-iT (I36933, Invitrogen) for 30 min prior to incubation with Phalloidin/Texas Red (T7471, Invitrogen) and Hoechst (B2261, Sigma-Aldrich) for 30 min. The coverslips were then mounted on microscope slides using Prolong Gold anti-fade reagent (P39634, Invitrogen). An inverted epifluorescence microscope (Axio Observer Z1, Carl Zeiss) equipped with an AxioCam MRm camera (Carl Zeiss) was used for fluorescence acquisition. Band pass filters with excitation at 560/40 nm and emission at 630/60 nm were used for Texas-Red (Chroma Technology Inc.). Band pass filters with excitation at 365/40 nm and emission at 445/50 nm were used for Hoechst (Carl Zeiss). Micrographs were acquired under a constant exposure time and presented under standardized luminosity and saturation attributes. Fluorescence quantification was achieved with ImageJ software [22 (link)]; see the Microscopy fluorescence image analysis section for additional details.
Hoechst
Manufactured by Zeiss
Sourced in Germany
The Hoechst is a precision laboratory equipment designed for a variety of applications. It functions as a versatile tool for conducting various analytical and research-based tasks within the controlled environment of a laboratory setting.
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Lab products found in correlation
Hoechst, by Thermo Fisher Scientific (5 mentions)
Lsm800 confocal microscope, by Zeiss (4 mentions)
Axio observer z1, by Zeiss (3 mentions)
Af488 conjugated goat anti mouse igg2b, by Thermo Fisher Scientific (2 mentions)
Pe anti ly 6g 1a8, by BioLegend (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Hoechst, by Merck Group (1 mentions)
Image it, by Thermo Fisher Scientific (1 mentions)
Texas red phalloidin, by Thermo Fisher Scientific (1 mentions)
Axiocam mrm camera, by Zeiss (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Mitogreen, by Thermo Fisher Scientific (1 mentions)
Zen blue image analysis software, by Zeiss (1 mentions)
Recombinant laminin 511, by BioLamina (1 mentions)
Nunc laboratory tek chambered coverglass, by Thermo Fisher Scientific (1 mentions)
Observer z1, by Zeiss (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Axiovert mrm, by Zeiss (1 mentions)
Mitosox red, by Zeiss (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
9 protocols using hoechst
1
Fluorescence Microscopy of AGE-HSA and CML-HSA Effects
2
Visualizing Mitochondrial Dynamics in Cardiomyocytes
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Three-hundred-thousand iCMs were seeded on a 29 mm glass-bottom (10 mm) dish (Cellvis, #D29-10-1.5-N, Sunnyvale, CA, USA). The following day, live cells were incubated with 0.5 µg/ml Mitogreen (#M7514, Thermo Fisher Scientific, Waltham, MA, USA) for mitochondria in medium at 37°C for 30 min. For NBT cells, 50,000 cells were seeded in an eight-chamber slide (#80826, Ibidi GmbH, Gräfelfing, Germany). The following day, live cells were incubated with 0.5 µg/ml Mitogreen (#M7514, Thermo Fisher Scientific) for mitochondria in medium at 37°C for 30 min together with Hoechst (#33342, Thermo Fisher Scientific) at 1 µg/ml final concentration. Images were taken by confocal microscopy (Zeiss LSM710, software Zen2.1) using excitation of 490 nm and emission of 516 nm for Mitogreen and excitation of 405 nm for Hoechst.
3
RBC Ca2+ Permeability Assay
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Fluo-4-stained RBCs (0.5 × 106; 1 μM; Invitrogen) were added to microscopic chambers (Nunc Laboratory-Tek Chambered Coverglass; ThermoFisher Scientific, Waltham, MA) coated with 0.5 μg recombinant laminin-511 (BioLamina, Sundyberg, Sweden) for 2 hours at 37°C. IRBCs were stained with 1 μg/mL Hoechst (Sigma, Spruce). Ionomycin (Sigma-Aldrich) treated RBCs (5 μM for 30 minutes at 37°C) were used as a positive control for increased intracellular Ca2+ levels. Ca2+ permeability was visualized with Observer Z1 (Zeiss, Oberkochen, Germany) taking frames of a defined area in the microscopic chamber every 5 minutes for 21 hours. Images were analyzed by ZEN blue image analysis software (Zeiss) to determine mean fluorescence intensities for Fluo-4 and for the quantification of translucent spherical Hoechst-negative uRBC ghosts.
4
Hoechst Staining of Cell Cultures
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After the culture period, the cells were washed with PBS and stained with Hoechst in the dark for 5 min. Photomicrographs were taken using a Zeiss inverted Axiovert CFL40 microscope equipped with a Zeiss Axiovert MRm monochrome camera using the following filters: Hoechst (Excitation: 352 nm, Emission: 455 nm) (Carl Zeiss, Oberkochen, Germany).
5
Quantification of Cellular and Mitochondrial ROS
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Total cellular ROS in cells was detected using the DCFDA/H2DCFDA-Cellular ROS kit from Abcam (ab11351) according to the manufacturer’s instructions. LNCaP and LN95 cells plated on 96-well, black, clear-bottom plates were stained with DCFDA/H2DCFDA (20 μM) for 30 min at 37 °C. After washing, cells were treated with tBHP (75 μM), enzalutamide (5 μM), ralaniten (35 μM), EPI-7170 (5 μM), or DMSO vehicle for 4 h. Fluorescence was measured using the Infinite M1000 (Tecan) with excitation at 485 nm and emission at 535 nm.
Mitochondrial superoxide was detected using MitoSOX Red from Sigma (M36008). LN95 cells were seeded on 8-well chamber slides and treated with tBHP, enzalutamide, ralaniten, EPI-7170, or DMSO vehicle as above for 4 h at 37 °C. Cells were then washed and stained with MitoSOX Red (5 μM) diluted in HBSS/Ca/Mg buffer (Gibco, 14025-092) for 10 min at 37 °C. Cells were washed gently three times in warm buffer before counterstaining with Hoechst 33342 (Sigma, 62249). Slides were mounted and visualized using a fluorescence microscope (Axio Imager.M2, ZEISS) at Ex/Em 350/461 (Hoechst) and 510/580 (MitoSOX Red).
Mitochondrial superoxide was detected using MitoSOX Red from Sigma (M36008). LN95 cells were seeded on 8-well chamber slides and treated with tBHP, enzalutamide, ralaniten, EPI-7170, or DMSO vehicle as above for 4 h at 37 °C. Cells were then washed and stained with MitoSOX Red (5 μM) diluted in HBSS/Ca/Mg buffer (Gibco, 14025-092) for 10 min at 37 °C. Cells were washed gently three times in warm buffer before counterstaining with Hoechst 33342 (Sigma, 62249). Slides were mounted and visualized using a fluorescence microscope (Axio Imager.M2, ZEISS) at Ex/Em 350/461 (Hoechst) and 510/580 (MitoSOX Red).
6
Visualizing Phagocytosis in SF Cells
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Paraffin embedded sections were visualized using FITC channel only by the Zeiss LSM800 confocal microscope. For evaluating phagocytosis, SF samples were initially collected as described above. SF cells were filtered and stained with PE-anti-Ly-6G (1A8, BioLegend) plus Hoechst (33342, Invitrogen, ThermoFisher Scientific) for 30 min at ambient temperature. After the staining, cells were fixed, permeabilized (BD Cytofix/CytoPerm solution, described above), and further stained intracellularly with AF488-conjugated goat anti-mouse IgG2b (polyclonal, ThermoFisher Scientific) in 1X Perm solution for 1 h. After sufficient wash by the same Perm solution, cells were finally visualized under the 100X objective of the Zeiss LSM800 system using AF488, PE, and Hoechst channels, with compensations been optimized.
7
Endosomal pH Neutralization Assay
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Endosomal pH neutralization was assayed essentially as described by Slater et al.58 (link); IMR90 cells in complete EMEM were plated at 14,000 cells/well (~95% confluency). After 24 h, the media was changed to SFM for 60 min, then compound was added to 40 μM and incubated at 37 °C for 2 h. LysoTracker red DND-99 and Hoechst (Life Technologies) were added to 0.1 and 1 μM, respectively, and incubated for 60 min. Excess dye was removed by media change and the fluorescence at ex/em 574/594 was read on an Envision plate reader (Perkin Elmer). Representative cell images were taken using a Zeiss Axiovert fluorescence microscope using DAPI and Texas Red filters to visualize the Hoechst and LysoTracker staining, respectively.
8
3D Melanoma Spheroid Cytotoxicity Assay
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Human SK‐MEL‐28 cells were seeded at a density of 1 × 104 cells per well in 96‐well ultralow attachment plates (PerkinElmer, Germany) and centrifuged at 1000 × g for 10 min. Consistent 3D melanoma spheroids formed after 48 h followed by drug, plasma, or combined treatment as described before. Spheroid cytotoxicity was evaluated using High Content Imaging (Operetta CLS; PerkinElmer, Germany) 24 h after exposure. Briefly, spheroids were stained with 5 µm SG for discrimination of terminally dead cells and 40 µm Hoechst (both Thermo Scientific, Germany), for nuclear staining, for 1 h at 37 °C. Immediately after, images were acquired in brightfield and fluorescence channels at λex 490 nm and λem 520 nm for SG and λex 365 nm and λem 465 nm for Hoechst from a z‐stack of 30 images at a 10 µm distance using a 5× air objective (NA = 0.16; Zeiss, Germany). Algorithm‐driven image analysis was performed using Harmony 4.9 image analysis software (PerkinElmer, Germany).
9
Visualizing Phagocytosis in SF Cells
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Paraffin embedded sections were visualized using FITC channel only by the Zeiss LSM800 confocal microscope. For evaluating phagocytosis, SF samples were initially collected as described above. SF cells were filtered and stained with PE-anti-Ly-6G (1A8, BioLegend) plus Hoechst (33342, Invitrogen, ThermoFisher Scientific) for 30 min at ambient temperature. After the staining, cells were fixed, permeabilized (BD Cytofix/CytoPerm solution, described above), and further stained intracellularly with AF488-conjugated goat anti-mouse IgG2b (polyclonal, ThermoFisher Scientific) in 1X Perm solution for 1 h. After sufficient wash by the same Perm solution, cells were finally visualized under the 100X objective of the Zeiss LSM800 system using AF488, PE, and Hoechst channels, with compensations been optimized.
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