OCCLs were plated on round glass coverslips (12 mm diameter) (150,000 cells/well in 6-well plates), and, after 24 h of culture, cells were submitted to different treatments, fixed with 4% paraformaldehyde for 10 min, and permeabilized using 0.5% Triton X-100 (Boehringer Mannheim, Mannheim, Germany) in PBS for 10 min. Then, samples were blocked in 10% BSA in PBS for 30 min and incubated with phospho-H2AX antibody (1:1000, 05-636, Sigma-Aldrich) for 90 min. After washing, coverslips were incubated with fluorescent secondary antibodies (1:400, Alexa Fluor 488 goat anti-mouse IgG, Molecular Probes, Invitrogen) for 1 h. DAPI (dihydrochloride of 4′,6-diamidino-2-phenylindole, Roche) was used to visualize the nuclei. Mowiol reagent (Calbiochem, San Diego, CA, USA) was used to fix preparations on slides. Cells were then analyzed by confocal microscopy (63×) using a LEICA SP5 microscope DMI-6000V model coupled to a Leica Application Suite X software computer (version 3.5.7.23225).
Sp5 microscope dmi 6000v model
Manufactured by Leica
The Leica SP5 microscope DMI-6000V model is a high-performance confocal laser scanning microscope. It features a flexible and modular design, allowing for customization to meet the specific needs of researchers and scientists. The SP5 microscope provides advanced imaging capabilities, including multi-channel fluorescence detection, high-resolution imaging, and the ability to perform time-lapse experiments.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (2 mentions)
Las af computer software, by Leica (2 mentions)
Triton x 100, by Roche (2 mentions)
Nb600 1384, by Novus Biologicals (1 mentions)
Alexa fluor 594 anti rabbit, by Thermo Fisher Scientific (1 mentions)
3 protocols using sp5 microscope dmi 6000v model
1
Immunofluorescence Analysis of DNA Damage
2
Immunofluorescence Analysis of DNA Damage
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OCCLs were plated on round glass coverslips (12 mm diameter) (250,000 cells/well in 6-well plates) and after 24 h of culture, cells were treated with Chloroquine, NAC and/or Panobinostat for 48 or 72 h. Then, cells were fixed with 4% paraformaldehyde for 10 min, permeabilized using 0.5% Triton X-100 (Boehringer Mannheim) in PBS for 10 min, blocked in 10% BSA in PBS for 30 min and incubated with phospho-H2AX antibody (1:1000, 05-636, Sigma-Aldrich) and/or Rad51 antibody (1:1000, PC130, Sigma-Aldrich) or LC3B (1:200, NB600-1384, Novus Biologicals) for 1h 30min. After washing, coverslips were incubated with fluorescent secondary antibodies (1:400, Alexa Fluor 488 goat antimouse IgG and/or Alexa Fluor 594 antirabbit, Molecular Probes, Invitrogen) for 1h. DAPI (dihydrochloride of 4’, 6-diamidino-2-phenylindole, Roche) was used to visualize the nuclei. Mowiol reagent (Calbiochem) was used to fix preparations on slides. Cells were then analyzed by confocal microscopy (63x) using a LEICA SP5 microscope DMI-6000V model coupled to a LEICA LAS AF software computer.
3
Quantifying DNA Damage in Cancer Cells
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Cancer cell lines were plated on round glass coverslips (12 mm diameter) (200,000 cells/well in 6-well plates) and, after 24 h of culture, cells were treated with CQ or CQ+NAC for 48 h. Then, cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X-100 (Boehringer Mannheim) in PBS for 10 min, blocked in 10% BSA in PBS for 30 min and incubated with phospho-H2AX antibody (1:1000, Sigma-Aldrich, RRID: AB_309864) for 90 min. After washing, coverslips were incubated with fluorescent secondary antibodies (1:400, Alexa Fluor 488 goat anti-mouse IgG, RRID: AB_141607) for 1 h. DAPI (dihydrochloride of 4’, 6-diamidino-2-phenylindole, Roche) was used to visualize the nuclei. Mowiol reagent (Calbiochem, San Diego, CA, USA) was used to fix preparations on slides. Cells were then analyzed by confocal microscopy (63x) using a LEICA SP5 microscope DMI-6000V model coupled to a LEICA LAS AF software computer.
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