HT-29 colorectal adenocarcinoma cells (2 × 105 cells/mL, 0.5 mL/well) were seeded on coverslips in 24-well cell culture plates and incubated for 24 h (37 °C, 5% CO2, 95% humidity) under cell culture conditions. After treatment with different concentrations of test compound 1a , 1b , 2 or the vehicle DMSO, the cells were incubated for another 24 h (37 °C, 5% CO2, 95% humidity). The cells were washed with cytoskeletal buffer (10 mM MES, 3 mM MgCl2, 138 mM KCl, 2 mM EGTA, pH 6.8), fixed and permeabilized in 3.7% formaldehyde and 0.2% Triton X-100 in cytoskeletal buffer for 5 min at rt. As additional fixation step, the cells were incubated with ice-cold EtOH for 10 s and rehydrated in PBS. Actin staining was done with Actistain 488 phallodin (100 nM in PBS) for 30 min at rt in the dark. Finally, the cells were washed three times with PBS and the coverslips were embedded in Roti®-Mount FluorCare (Roth) with 1 µg/mL DAPI for nuclei staining. Actin filaments and nuclei were documented by confocal microscopy (Leica Confocal TCS SP5, 630× magnification).
Confocal tcs sp5
Manufactured by Leica
Sourced in Germany, United States
The Confocal TCS SP5 is a high-performance confocal laser scanning microscope designed for advanced imaging applications. It features a modular design that allows for flexible configuration and customization to meet the specific needs of researchers and scientists. The system utilizes state-of-the-art laser technology and optical components to provide high-resolution, high-sensitivity, and high-speed imaging capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Superfrost plus slide, by Thermo Fisher Scientific (2 mentions)
Roti mount fluorcare, by Carl Roth (1 mentions)
Alexa fluor 594 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Confocal tcs sp8, by Leica (1 mentions)
Formamide, by Avantor (1 mentions)
Axiovision 4, by Zeiss (1 mentions)
Metamorph nx, by Molecular Devices (1 mentions)
Axio imager z1 fluorescent microscope, by Zeiss (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Imagej, by Adobe (1 mentions)
5 protocols using confocal tcs sp5
1
Imaging Actin Cytoskeleton of HT-29 Cells
2
Visualizing Actin Cytoskeleton in Saos-2 Cells
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For further cell adhesion examination, cell cultures were rinsed twice with PBS and fixed with 4% paraformaldehyde in PBS for 30 min at RT, then rinsed twice in PBS. Cells were permeabilized with 0.1% triton X-100 in PBS for 15 min at RT. Actin filaments were stained by incubating the samples with Alexa Fluor 594-conjugated phalloidin (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA), and the cell nucleus was stained with Hoechst 33,258 (Sigma-Aldrich, Saint Louis, MO, USA) for 15 min at RT in the dark. Saos-2 actin distribution was observed under a confocal microscope (Confocal TCS SP5, Leica, Wetzlar, Germany).
3
Immuno-FISH Analysis of DNA Damage Markers
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Immuno-FISH was performed as described. [50 (link)] Briefly, cells were harvested and cytospined onto Superfrost plus slides (Thermo Scientific), fixed with 4% paraformadehyde and permeabilized with Triton PBS for 20 mins and blocked with serum free Block (DAKO, Glostrup, Denmark). The slides were then incubated with 53BP1 antibody (Bethyl Inc., Montgomery, Texas, USA) followed by incubation with Alexa 594 secondary antibody (Jackson Labs Technologies Inc., Los Gatos, CA, USA). The slides were treated with frozen and thawed cycle in liquid nitrogen, and incubated in 0.1N HCL for 10 mins. The PNA-telomere probe (PANAGENE Inc., Daejeon, Korea) was finally added. High-resolution images were collected using Leica Confocal TCS SP5 with 488 nm and 594 nm sequential laser scan. Depth of Z stack was taken with recommended optimization and between-stack mode. The co-localization of 53BP1 and telomere signals was examined and analyzed on each layer by two separate independent persons in a double-blind manner. The detailed protocol is available upon request.
4
Confocal Imaging of Fluorescent Proteins
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Imaging was performed by using a Confocal TCS SP8 and a Confocal TCS SP5 (Leica) equipped with PMT detectors both for fluorescence and transmitted light images. AOBS beam-splitter systems are in place on both instruments. 63X oil immersion objectives and the LAS AF software were used for all imaging. YFP fluorescence was excited with a 488 nm laser, while mCherry fluorescence with a 561 nm laser. Ranges for emission detection were 495–554 and 637–670 nm respectively. Image depth is 8-bit in all cases and pixel size equals 120.4 nm. The LUT is linear and covers the full range of the data.
5
Cytosolic dsDNA and DNA-RNA Hybrid Staining
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Cytosolic dsDNA and DNA-RNA staining were performed as described previously (16). Briefly, 50% formamide (VWR International, USA) diluted in PBS was added for 10 mins at room temperature, followed by incubation for 15 mins at 75°C to denature dsDNA. Cells were washed with PBS and incubated with blocking buffer (1% BSA (Sigma Aldrich), 2% goat serum (Hyclone) in PBS) for 1 h to prevent non-specific binding of antibodies. Cells were then stained with dsDNA (1:200, Sigma-Aldrich, USA) or S9.6 DNA-RNA hybrids (1:100, Kerafast, Boston, USA) antibodies overnight in 4°C. Cells were subsequently washed with PBST (0.1% Tween) thrice followed by anti-mouse IgG coupled with Cy3 (Millipore, Singapore) or anti-rabbit IgG coupled to Alexa Fluor 488 (Invitrogen, Singapore). Finally, DNA fluorophore DAPI (0.5 μg/ml in PBS, KPL Inc., USA) was added for 10 min. Slides were washed once in PBS before mounted with Da-/- fluorescent mounting medium (Da-/-, UK). Confocal images of staining were captured using a Zeiss Axio Imager Z1 fluorescent microscope equipped with AxioVision 4.8 software (Carl Zeiss MicroImaging, USA) or confocal TCS SP5 (Leica, Singapore). Images were analyzed using Photoshop CS4 (Adobe, USA) or ImageJ. Colocalisation of AIM2 with DNA or other proteins were quantified using Metamorph (Metamorph NX, version 8.12, Molecular Devices, USA).
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