Cells were stained with 5 μM SYTOXGreen (SG) (Life Technologies) and cell death measured with an Incucyte ZOOM system (Essen BioScience, Michigan, USA). Percent cell death was calculated as follows: [100 × (inducedfluorescence − backgroundfluorescence)] ÷ (maximalfluorescence achieved by Triton-X-100 permeabilization − backgroundfluorescence). The data are presented as mean ± standard error of the mean (S.E.M) of at least 2–3 independent experiments. All cell death profiles in this paper are shown as mean ± S.E.M. For other statistical analysis, two-tailed unpaired T-test was used. *P < 0.05; **P < 0.01; ***P < 0.001 were considered as significant.
Sytox green sg
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
SYTOX green (SG) is a nucleic acid stain used for the detection of dead cells. It is a cell-impermeable dye that enters cells only with compromised membranes, and then binds to nucleic acids, resulting in bright green fluorescence. The core function of SYTOX green is to provide a reliable indicator of cell viability.
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Lab products found in correlation
Incucyte zoom system, by Sartorius (1 mentions)
Fluostar omega, by BMG Labtech (1 mentions)
Vybrant did cell labeling solution, by Thermo Fisher Scientific (1 mentions)
Flat bottom 96 well plate, by Greiner (1 mentions)
96 well ultra low attachment plate, by Thermo Fisher Scientific (1 mentions)
Harmony 4, by PerkinElmer (1 mentions)
Ckx53, by Olympus (1 mentions)
6 protocols using sytox green sg
1
SYTOX Green Cell Death Assay
2
Measuring Cell Death and Caspase-3 Activity
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Cell death and caspase-3 like activity analysis using the Fluostar Omega fluorescent plate reader (BMG Labtech GmbH, Ortenberg, Germany) was performed as previously described.33 (link) Briefly, the cells were seeded in triplicate at 7500 cells/well in a 96-well plate. The next day the cells were treated with the indicated compounds in the presence of 5 μM SYTOX Green (SG) (Life Technologies, Carlsbad, CA, USA) and for some experiments in combination with 20 μM DEVD-AMC (PeptaNova GmbH, Sandhausen, Germany). SG and AMC fluorescence intensity were measured in function of time with excitation/emission filters of 485/520 nm and 360/460 nm, respectively. The percentage of cell death was calculated as (induced SG fluorescence−background SG fluorescence)/(maximal SG fluorescence−background SG fluorescence)X100. The maximal SG fluorescence was obtained by full permeabilization of the cells using 0.05% Triton X-100. Caspase-3like activity was calculated as (induced AMC fluorescence−background AMC fluorescence).
3
Enhancing DNA Tethering via Surface Passivation
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First, the channel
of the flowcell
was filled with buffer B. To enhance surface passivation against nonspecific
protein adsorption, we injected 5% Tween-20 solution in buffer B into
the channel of the flowcell, incubated for 10 min, and washed out
with 600 μL of buffer A.28 (link) Next,
the biotinylated DNA (∼30 pM, in buffer B) was added and incubated
for at least 15 min. The excess of unbound DNA was washed out with
∼300 μL of buffer A. Then, DNA was labeled with the DNA
intercalating green fluorescent dye, SYTOX green (SG, ThermoFisher
Scientific), at a concentration of ∼0.4 nM (in imaging buffer:
buffer A supplemented with 0.2% Tween-20, 1 mM dithiothreitol, DTT).
The SG dye was present during the entire time of the experiment. In
the case of the second DNA end tethering, the close-loop circulation
was employed and 5 μL of biotin-anti-dig (bt-anti-dig) antibody
was added (this resulted in ∼0.05 mg/mL concentration) and
incubated for at least 10 min at low speed (∼0.1 mL/min). After
10 min, the speed of the buffer flow was increased to ∼1 mL/min
and kept constant for 20 min. Then, to remove the excess of unbound
bt-anti-dig, the flowcell was washed with 500 μL of buffer A
in the open-loop circulation. Finally, 100 μL of imaging buffer
was injected into the flowcell to reveal bound DNA.
of the flowcell
was filled with buffer B. To enhance surface passivation against nonspecific
protein adsorption, we injected 5% Tween-20 solution in buffer B into
the channel of the flowcell, incubated for 10 min, and washed out
with 600 μL of buffer A.28 (link) Next,
the biotinylated DNA (∼30 pM, in buffer B) was added and incubated
for at least 15 min. The excess of unbound DNA was washed out with
∼300 μL of buffer A. Then, DNA was labeled with the DNA
intercalating green fluorescent dye, SYTOX green (SG, ThermoFisher
Scientific), at a concentration of ∼0.4 nM (in imaging buffer:
buffer A supplemented with 0.2% Tween-20, 1 mM dithiothreitol, DTT).
The SG dye was present during the entire time of the experiment. In
the case of the second DNA end tethering, the close-loop circulation
was employed and 5 μL of biotin-anti-dig (bt-anti-dig) antibody
was added (this resulted in ∼0.05 mg/mL concentration) and
incubated for at least 10 min at low speed (∼0.1 mL/min). After
10 min, the speed of the buffer flow was increased to ∼1 mL/min
and kept constant for 20 min. Then, to remove the excess of unbound
bt-anti-dig, the flowcell was washed with 500 μL of buffer A
in the open-loop circulation. Finally, 100 μL of imaging buffer
was injected into the flowcell to reveal bound DNA.
4
3D Hydrogel Cultures of Labeled Cells
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For the prior generation of the 3D hydrogel cultures, the cells were stained with 500 nM Vybrant DiD cell labeling solution (Thermo Fisher Scientific, Dreieich, Germany) for 45 min at 37 °C. After washing, the cells were resuspended in 4 mg/mL collagen I (Enzo Life Sciences, Lörrach, Germany) containing 1% sodium bicarbonate (NaHCO3) and 19% 10× Minimal Essential Medium (MEM; Corning, Kaiserslautern, Germany), as described above. Additionally, 500 nM sytox green (SG; Thermo Fisher Scientific, Dreieich, Germany) was added for the live–dead cell discrimination. The cells were seeded in a 96-well flat-bottom plate (Greiner Bio-One, Frickenhausen, Germany) at a density of 2 × 105 cells in 100 µL collagen per well and incubated for 1 h at 37 °C to induce hydrogel polymerization. In parallel, the cells were seeded in cell culture medium at a density of 1 × 104 cells per well to compare the cellular growth in conventional 2D and 3D hydrogel cultures. In the 2D experiments, the number of planted cells was limited to 1 × 104, as they would overgrow and consume nutrients during the growth period at higher seeding densities. The cell lines were randomly chosen in an in-house screening.
5
Gas Plasma Treatment of 3D Urothelial Carcinoma Spheroids
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Three-dimensional (3D) urothelial carcinoma spheroids were prepared by seeding 2x104 cells per well in a 96-well ultra-low attachment plate (Thermo Fisher, Germany) and following centrifugation at 1000xg for 10 min. Cells were incubated for two days to allow spheroid formation. After 48 h, spheroids were exposed to gas plasma for 30 s, 60 s, or 90 s or argon gas only as mock control. Immediately after gas plasma treatment, spheroids were stained with 0.1 µM Sytox Green (SG; Thermo Fisher, Germany) to label terminally dead cells. Evaluation of gas plasma-mediated tumor toxicity was done using fluorescence imaging 0 h, 24 h, and 48 h after treatment. Images were acquired in brightfield and fluorescence channels (λex 475 nm and λem 525 nm for SG) with a 5x air objective (NA = 0.16) and ten z-stacks per well and spheroid. The brightfield signal was inverted for image analysis, and an intensity cut-off was defined to distinguish the spheroid region from the background. In this area, the mean fluorescence intensity (MFI) of SG was quantified. Image acquisition parameters and unsupervised algorithm-based image analysis were performed using Harmony 4.9 software (PerkinElmer, Germany).
6
Bamboo Bud Morphology Imaging
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Dormant and active lateral buds of 11 bamboo species were sectioned longitudinally and stained with Sytox green (SG, Thermo Fisher Scientific) to monitor mitotic activity and counterstained with Safranin O (SF, Wako Pure Chemical Corp.) to detect the inward region of the SAM region based on a previous method [16] (link) with a minor modification.
In order to evaluate growth performance of explants, whole images of in vitro cultured shoots were captured with a digital camera system (UV CUBE, LC science Co., Ltd.) under a bright-field and LED 365 nm illumination without staining. The autofluorescence property of the obtained images was evaluated by RGB (red, green, and blue) digital imaging analysis using ImageJ software. A stereo microscope (SZ40, Olympus) was also used to monitor the morphological characteristics of the target tissue. The detailed autofluorescence properties of the outward regions of culm and node tissues were observed using an inverted cell culture microscope (CKX53, Olympus) under B-and U-excitation lights [B; Band pass (BP) filter, 460 -495; Barrier (BA) filter, 510IF; Dichroic mirror (DM), 505, U-FUW; BP filter, 340 -390; BA filter, 420IF; DM, 410] with RGB and HSB (hue, saturation, and brightness) digital imaging analysis. The HSB color space in ImageJ software is regarded as equivalent to the HSV (hue, saturation, and value) color space.
In order to evaluate growth performance of explants, whole images of in vitro cultured shoots were captured with a digital camera system (UV CUBE, LC science Co., Ltd.) under a bright-field and LED 365 nm illumination without staining. The autofluorescence property of the obtained images was evaluated by RGB (red, green, and blue) digital imaging analysis using ImageJ software. A stereo microscope (SZ40, Olympus) was also used to monitor the morphological characteristics of the target tissue. The detailed autofluorescence properties of the outward regions of culm and node tissues were observed using an inverted cell culture microscope (CKX53, Olympus) under B-and U-excitation lights [B; Band pass (BP) filter, 460 -495; Barrier (BA) filter, 510IF; Dichroic mirror (DM), 505, U-FUW; BP filter, 340 -390; BA filter, 420IF; DM, 410] with RGB and HSB (hue, saturation, and brightness) digital imaging analysis. The HSB color space in ImageJ software is regarded as equivalent to the HSV (hue, saturation, and value) color space.
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