Coverslips were sterilized in ethanol, and cells were plated on coverslips in 12-well plates. Cells were allowed to adhere overnight before treatment with drug-containing media for one hour prior to RT (4 Gy). Following RT, cells were grown until designated time points and fixed using 4% paraformaldehyde (Thermo Scientific J19943K2). Cells were blocked in a solution containing goat serum (ThermoFisher 16210064) and stained using an anti-Rad51 antibody (GeneTex GTX70230, 1:300) and a fluorescent goat anti-mouse secondary antibody (Invitrogen A11005, 1:2000). Nuclei were stained with ProLong Gold antifade reagent with DAPI (Invitrogen P36931). Pictures of >100 cells were taken using a Nikon Fluorescent Microscope. Cells with ≥10 Rad51 foci/cell were counted as positive. Results were pooled for statistical analyses, and data are shown as the mean ± SD for three or four replicate experiments.
Gtx70230
Manufactured by GeneTex
GTX70230 is a laboratory microcentrifuge designed for the separation of samples. It has a maximum speed of 15,000 RPM and a maximum relative centrifugal force of 21,380 x g. The microcentrifuge can accommodate sample volumes up to 2 mL.
Automatically generated - may contain errors
Lab products found in correlation
A11005, by Thermo Fisher Scientific (2 mentions)
Fluorescent microscope, by Nikon (1 mentions)
P36931, by Thermo Fisher Scientific (1 mentions)
Dm 6000rxa fluorescence microscope, by Leica (1 mentions)
Application suite software, by Leica (1 mentions)
Ab79398, by Abcam (1 mentions)
Deltavision deconvolution microscope, by GE Healthcare (1 mentions)
Prolong glass anti fade medium, by Thermo Fisher Scientific (1 mentions)
Quasar 570 fluorophore, by Biosearch Technologies (1 mentions)
Nucblue nuclear counterstain, by Thermo Fisher Scientific (1 mentions)
Stellaris rna fish probe set, by Biosearch Technologies (1 mentions)
S9378, by Merck Group (1 mentions)
4 protocols using gtx70230
1
Rad51 Foci Quantification Protocol
2
Ionizing Radiation-Induced RAD51 Foci Analysis
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Cells were left untreated or were irradiated with 10Gy using a Cesium137 source (CIS international/IBL 637 irradiator, dose rate: 0.01083 Gy/s). Three hours later, cells were fixed in 2% paraformaldehyde with 0.1% Triton X-100 in PBS for 30 minutes at room temperature. Cells were permeabilized in 0.5% Triton X-100 in PBS for 10 minutes. To block nonspecific binding, cells were incubated with PBS containing 0.05% Tween-20 and 4% BSA (Fraction V) (PBS-Tween-BSA) for 1 hour. Cells were incubated overnight at 4°C with mouse anti-RAD51 (GeneTex, GTX70230, 1:400) in PBS-Tween-BSA. Cells were extensively washed and incubated for 45 minutes with Alexa488-conjugated secondary antibodies. Images were acquired on a Leica DM-6000RXA fluorescence microscope, equipped with Leica Application Suite software.
3
RNA FISH and Immunofluorescence Analysis
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For RNA FISH, 5 days post-transduction, CRISPRa and Tet-on KILR overexpressing cells grown on coverslips were fixed in 4% formaldehyde for 10 min followed by permeabilization in 70% ethanol overnight at 4 °C. Cells were then stained for 16 h with 125 nM of a custom KILR Stellaris RNA-FISH probe set labelled with Quasar 570 fluorophore (LGC Biosearch Technologies) according to the manufacturer’s instructions. For IF, CRISPRa, CRISPR-Cas13, Tet-On and RPA1-KILR overexpressing cells were challenged with or without 6 Gy gamma irradiation followed by 6 h of incubation. The cells were then treated with CSK buffer (10 mM PIPES, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1.4% Triton X-100) to remove the cytoplasm, followed by fixation in 4% formaldehyde for 10 min and permeabilization with 0.5% Triton X-100 for 15 min. The cells were incubated with antibodies against RPA1/RPA70 (Abcam, ab79398, 1:250), γH2AX (Abcam, ab2893, 1:1000) or RAD51 (GeneTex, GTX70230, 1:500). Coverslips were mounted onto slides using ProLong Glass antifade medium containing NucBlue nuclear counterstain (Thermo Fisher Scientific). Images were acquired with the DeltaVision Deconvolution microscope (GE Healthcare) using a 60x objective lens and analyzed with ImageJ software. A minimum of 100 cells per sample were analyzed.
4
Radiation-Induced DNA Damage and Repair
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A total of 100,000 cells was plated onto coverslips in 12-well plates and treated the next morning with either palbociclib or vehicle (DMSO) 1 hour prior to radiation (4 Gy). Coverslips were fixed at 6 hours or 16 hours after RT (4 Gy) for RAD51, and γH2AX foci were fixed at 0.5, 6, 16, and 24 hours after RT (2 Gy). Cells were fixed in 4% paraformaldehyde (Thermo Fisher Scientific, J19943K2) with 2% sucrose (MilliporeSigma, S9378) and 0.2% Triton X-100 (MilliporeSigma, T8532), permeabilized with 0.5% Triton X-100, and blocked in 1× PBS containing 5% goat serum (Thermo Fisher Scientific, 16210064), 0.5% BSA, and 0.05% Triton X-100. The phospho-histone H2AX (S139) monoclonal antibody (MilliporeSigma, 05-636, 1:2000) or the anti-RAD51 antibody (GeneTex, GTX70230 1:300) were used with the goat anti–mouse fluorescent secondary antibody (Invitrogen, A11005, 1:2000) to stain foci. A minimum of 100 cells were used to score and analyze formation of γH2AX and RAD51 foci. To quantify the exact number of foci per cell, ImageJ (NIH) was used to count image maxima. Cells with more than 15 γH2AX foci or more than 10 RAD51 foci were scored as positive. To quantify micronuclei formation, cells were radiated at 4 Gy and fixed in 4% paraformaldehyde after 72 hours before mounting directly on coverslips with DAPI stain (34 (link)).
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