Biofilms were grown in 96-well plates (μclear, Greiner Bio-One). A total of 150 µL of BHIS, supplemented with 0.5% bile extract, 25 µg/mL of PAßN, or 35 mM magnesium when required, was added to each well, and the plates were incubated at 37°C, in static condition 48 h under anaerobic conditions. The unwashed biofilms were then directly stained in red with 5 µM of SYTO61 (Life Technologies; cell permeant nucleic acid dye to contrast all the bacteria). After 15 min of incubation, Z stacks of horizontal plane images were acquired in 1 µm steps using CLSM (Leica TCS SP8, INRAE MIMA2 microscopy platform) with a water 63 × immersion lens [numerical aperture (NA) = 1.2]. Two stacks of images were acquired randomly on three independent samples at 800 Hz. Fluorophores were excited, and emissions were captured as prescribed by the manufacturer. Simulated 3D fluorescence projections were generated using IMARIS 9.3 software (Bitplane). Biofilm biovolumes (µm3) extracted from CLSM images were analyzed with BiofilmQ (48 (link)).
μclear
Manufactured by Greiner
Sourced in Germany
μClear is a specialized laboratory equipment designed for cellular imaging. It provides high-quality, transparent, and uniform cell culture plates that enhance microscopic observation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (8 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Lsm800 confocal microscope, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Syto 61, by Thermo Fisher Scientific (1 mentions)
Tcs sp8, by Leica camera (1 mentions)
Accutase, by Thermo Fisher Scientific (1 mentions)
Alexa 647, by Thermo Fisher Scientific (1 mentions)
Harmony software, by PerkinElmer (1 mentions)
Resazurin, by R&D Systems (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Opera high content screening system, by PerkinElmer (1 mentions)
Lsm 800 microscope, by Zeiss (1 mentions)
α actinin, by Merck Group (1 mentions)
Mouse alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Histofix, by Carl Roth (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
Triton x 100, by Carl Roth (1 mentions)
32 protocols using μclear
1
Quantitative Biofilm Analysis by CLSM
2
Fibrinogen Hydrogel Mechanical Properties
3
Optimization of Escherichia coli Cultivation
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For the investigations in dataset A and C-F, Escherichia coli Tuner(DE3)/pRhotHi-2-LacI-EcFbFP was cultivated in 48-well FlowerPlates (MTP-48-B, lot 1404 & 1509, m2p-labs, Baesweiler, Germany). Two pre-cultures (first: complex TB medium; second: synthetic Wilms-MOPS medium) were conducted in 250 mL shake flask (37 °C; shaking frequency (n): 350 rpm; shaking diameter (d0): 50 mm; filling volume (VL): 10 mL). Synthetic modified Wilms-MOPS medium with 20 g L−1 glucose was used for main culture. Further details about the media composition is given elsewhere [33 (link), 34 (link)].
In dataset G, cultures of Escherichia coli BL21-Gold (DE3) pET-t7-CelA2 were investigated. Again, two pre-cultures (first complex, second synthetic medium) and synthetic modified Wilms-MOPS medium with 20 g L−1 glucose for main culture were used. The time of induction was chosen between 1 and 10 h with IPTG concentrations between 50 and 1000 μM.
In dataset B Escherichia coli BL21(DE3) pRhotHi-2-EcFbFP was investigated. Synthetic MDG mineral medium [35 (link)] with 5 g L−1 glucose as carbon source was used for the main cultures in a 96-well MTP (μClear, Greiner Bio-One, Frickenhausen, Germany). Further information are given elsewhere [10 (link)].
In dataset G, cultures of Escherichia coli BL21-Gold (DE3) pET-t7-CelA2 were investigated. Again, two pre-cultures (first complex, second synthetic medium) and synthetic modified Wilms-MOPS medium with 20 g L−1 glucose for main culture were used. The time of induction was chosen between 1 and 10 h with IPTG concentrations between 50 and 1000 μM.
In dataset B Escherichia coli BL21(DE3) pRhotHi-2-EcFbFP was investigated. Synthetic MDG mineral medium [35 (link)] with 5 g L−1 glucose as carbon source was used for the main cultures in a 96-well MTP (μClear, Greiner Bio-One, Frickenhausen, Germany). Further information are given elsewhere [10 (link)].
4
Metabolic Modulation of Cell Culture
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For the experiments, cells were used from culture flasks at 70 to 90% confluency. Cells were first washed with 6 ml of Hanks (without Ca2+ / Mg2+: 137 mM NaCl; 5.4 mM KCl; 0.33 mM Na2HPO4× 2 H20; 0.44 mM KH2PO4; 2 mM Hepes (pH 7.4)) and then detached for 5 min in an incubator at 37°C using 2 ml Accutase (Life Technologies). Then, pre-warmed SCM (4 ml) was added and the cells were transferred to a centrifuge tube and collected by centrifugation (5 min; 125 × g). After aspiration of old medium, fresh SCM was added and 0.5x106 cells were equally spread in 10 ml of SCM into new culture flasks (250 ml). After 72 hours cells were again harvested as described above and seeded at a density of 5000 cells per well in 96-well plates (μClear, Greiner Bio One, Frickenhausen, Germany) in SCM for 3 hours before they received fresh medium without glucose, galactose or pyruvate and without GlutaMax and FBS. Then cells were incubated for 20 or 24 hours (see individual experiment) before they received new medium with the supplements indicated in each experiment.
5
Quantify DNA Methylation in ESCs
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ESCs were grown in 96-well microplates (μClear, Greiner Bio-One), washed with PBS, and fixed with 3.7% formaldehyde. After three washing steps with PBST, cells were permeabilized (0.5 % Triton-X100), treated with denaturing solution (2 N HCl) for 40 min, and incubated with renaturing solution (150 mM Tris-HCl, pH 8.5) for 20 min. Cells were then blocked in 2% bovine serum albumin for 1 h and incubated with primary antibody (mouse-anti 5mC, Diagenode 33D3) for 1 h at 37 °C. After washing three times with PBST, cells were incubated with secondary antibody (goat-anti-mouse coupled to Alexa647, Thermo Fisher) for 1 h at 37 °C. Cells were washed three times with PBST, counterstained with 200 ng/ml 4,6-diamidino-2-phenylindole (DAPI), and finally covered with PBS. Images were acquired by automation with an Operetta High-Content Image Analysis System (PerkinElmer, ×40 high NA objective) followed by analysis with the Harmony software (PerkinElmer). DAPI was used for the detection of single nuclei and 5mC modifications were measured in selected nuclei based on the antibody signal intensity.
6
Evaluating CML Cell Proliferation Inhibition
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CML cell proliferation in vitro was evaluated by the AlamarBlue assay using the NCI 60 human tumor cell line anticancer drug screen (NCI60) methodology that yielded growth inhibition 50% (GI50) values as a quantitative measure of viability (24 (link)). Three different batches of cells/line were harvested and plated in 384-well plates (Greiner μClear) at 1,300 cells per well and incubated for 24 hours at 37°C. The next day, all the compounds were screened in at three-fold dilution series, with four technical replicates and incubated for 48 hours at 37°C. Maximum doses of ponatinib, 33a, 36a, and nilotinib were 10 μmol/L for the K562 cell line and 1.11 μmol/L for the KCL22 cell line. At this point, the cells were treated with Resazurin (final concentration 10%; R&D Systems #AR002) and incubated for 2 hours before measuring fluorescence on a plate reader (excitation 544 nm, emission 590 nm) to quantify the antiproliferative effects of the compounds.
7
Quantitative Immunofluorescence Assay of eIF2α Phosphorylation
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For eIF2α phosphorylation staining, CT26 cells were seeded onto 384 well cell culture microplates, μClear® (Greiner bio-one, Kremsmünster, Austria), allowed to adapt for 48 h and then fixed with paraformaldehyde (PFA) for 30 min and stored at 4°C in PBS. To assess the staining, cells were washed with PBS, permeabilized with 0.3% Triton X-100 for 10 min at room temperature, RT, and rinsed three times with PBS. Non-specific binding sites were blocked with bovine serum albumin (BSA) for 15 min at RT followed by incubation with the primary antibody (1.4 mg mL−1) for 2 h at 37°C. Subsequently, cells were washed three times with PBS and incubated for 30 min in AlexaFluor® 488-conjugated secondary antibody (1:500 in BSA; Molecular Probes-Invitrogen, Eugene, USA). When appropriate, 10 μM Hoechst 33342 (Molecular Probes-Invitrogen) was used for nuclear counterstaining. Images were acquired using an Image Xpress Micro XLS high content imager (Molecular Devices (MDS), Sunnyvale, USA). Images were analyzed with the MetaXpress® software (MDS Analytical Technologies, Sunnyvale, USA).
8
Immunofluorescence Analysis of hiPSC-CMs
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Human iPSC-CMs were cultured for 7 days in 96-well plates (μclear, Greiner), then rinsed once with pre-warmed 1× PBS and fixed with Histofix (Carl Roth) for 20 min at 4°C. After washing two times in cold 1× PBS, hiPSC-CMs were incubated with primary antibodies directed against the M-motif of cMyBP-C (1:200, custom made), FLAG (1:800, Sigma), and α-actinin (1:800, Sigma); diluted in permeabilization buffer (1× PBS [Gibco], milk powder 3% [w/v, Carl Roth], and Triton X-100 0.1% [Carl Roth]); and incubated overnight at 4°C under gentle agitation. After washing two times in cold 1× PBS, hiPSC-CMs were incubated with secondary antibodies anti-mouse Alexa Fluor 488 (1:800, Life Technologies) and anti-rabbit Alexa Fluor 546 (1:800, Life Technologies), diluted in permeabilization buffer, and incubated for 1–2 hr at room temperature under gentle agitation and protected from light. In a final step, Hoechst 33342 (1:2,500, Thermo Fisher Scientific) diluted in 1× PBS was added to the wells and incubated for an additional 20 min. After washing two times in 1× PBS, hiPSC-CMs were ready for subsequent analysis in the Opera High-Content Screening System (PerkinElmer) or by confocal microscopy using a Zeiss LSM 800 microscope with Airyscan technology.
9
Mitochondrial Staining in Cells
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Cells were plated in clear bottom 96-well plates (μClear, Greiner) and treated as described in the legend of the figure. The cells were then incubated with 100 nM MitoTracker Deep Red FM (Thermo Fisher Scientific) for 30 min, at 37 °C. The medium was removed, and 1 μg/ml Hoechst 33342 was added for 15 min, at 37 °C. The solution was removed and cells were washed with PBS and fixed with 4% neutral-buffered formalin for 10 min, at room temperature. The MitoTracker Deep Red FM fluorescence signal was acquired at 644/665 nm (Ex/Em) and 350/461 nm (Ex/Em) for Hoechst 33342. The mitochondria staining was visualized in a fluorescence microscope at the same wavelengths.
10
High-Content Screening of 5-FU Effects
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For quantitative high-content screening (HCS) microscopy analyses cells were plated at a density of 2000 cells/well in a 96-well imaging plate (Greiner μClear) and treated with a concentration gradient of 5-FU as indicated. Cells were immunolabeled as described above and nuclei stained with DAPI (diamidinophenylindole, Sigma). Plates were scanned using a Thermo Fisher Cellomics ArrayScan VTI and images of 512 × 512 pixels were acquired with a 20x objective and analyzed using the Cellomics software package (Colocalization V.4 Bioapplication). Cell nuclei were identified by DAPI staining and according to the object identification parameters size: 100–1200 μm2, ratio of perimeter squared to 4π area: 1–2, length-to-width ratio: 1–2, average intensity: 50–1000, total intensity: 3×104–2×107. SGs and P-bodies were identified within a circular region extending the nucleus by maximally 20 μm. The object identification parameters for SGs and P-bodies were 1.5–20 μm2, ratio of perimeter squared to 4π area: 1–1.8, length-to-width ratio: 1–1.8, average intensity: 100–1500 and total intensity: 5×103–5×104.
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