Cells were cultured on multi Nunc Lab-Tek II chambered slides (Thermo Fisher Scientific) for 24 h, after which they were exposed to 1 Gy of irradiation in a CellRad system (Precision X-Ray). Cells were fixated at various time points after irradiation (30 min–24 h) in 4% paraformaldehyde for 10 min and subsequently permeabilized in PBS with 0.1% triton-X for 15 min. Non-specific adhesion sites were blocked for 45 min in 0.25% Tween-20/PBS with 1% bovine serum albumin, followed by the addition of primary antibodies against γ-H2AX (1:10.000, 05-636, Millipore) or RAD51 (1:50, PA5-27195, Thermo Fisher Scientific), or isotype immunoglobulin G in the case of negative controls. After overnight incubation at 4 °C, the cells were washed and incubated with the corresponding host-specific secondary antibodies goat anti mouse Alexa Fluor 488 (1:1.000, A11029, Thermo Fisher Scientific) or goat anti Rabbit Alexa Fluor 532 (1:1000, A11009, Life Technologies), and counterstained with DAPI. The slides were mounted with Prolong Gold anti-fade (Thermo Fisher Scientific) and visualized using a Leica DM5000B microscope. γ-H2AX and RAD51 foci within the cell nucleus were counted manually in at least 100 cells per condition. This was repeated 3 times for statistical analysis.
Pa5 27195
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PA5-27195 is a laboratory equipment product manufactured by Thermo Fisher Scientific. It serves as a core piece of equipment for various scientific applications. Its primary function is to perform a specific task within the laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
710 confocal microscope, by Zeiss (3 mentions)
A11029, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 532, by Thermo Fisher Scientific (1 mentions)
Ab128171, by Abcam (1 mentions)
Ab193353, by Abcam (1 mentions)
Fl 335, by Santa Cruz Biotechnology (1 mentions)
T9026, by Merck Group (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Qubit protein assay kit, by Thermo Fisher Scientific (1 mentions)
Deconvolution software, by Cytiva (1 mentions)
Coolsnaphq2, by Universal Imaging (1 mentions)
Metamorph software, by Universal Imaging (1 mentions)
Ab52866, by Abcam (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Sc 126, by Santa Cruz Biotechnology (1 mentions)
Jbw301, by Merck Group (1 mentions)
P0448, by Agilent Technologies (1 mentions)
Trans blot sd cell, by Bio-Rad (1 mentions)
Pierce bca assay kit, by Thermo Fisher Scientific (1 mentions)
6 protocols using pa5 27195
1
Quantifying DNA Damage Response
2
Quantifying Protein Expression via Western Blot
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Cells were seeded in 6-well plates. Once 80% confluency was reached, media was discarded and cells were washed twice with phosphate-buffered saline. Cells were treated with 1 mL 0.25% trypsin and incubated for 5 min, after which the corresponding media was added to dilute the trypsin. The mixture was pipetted from the plates to tubes. Proteins were extracted from the cells using a RIPA buffer and the addition of protease- and phosphatase-inhibitors. Protein expression was quantified with the Qubit Protein Assay Kit (Thermo Fisher Scientific). Western blot analysis was performed using the LI-COR Odyssey imaging system (LI-COR Biosciences) as previously reported [19 (link)]. The primary antibodies were mouse anti-TRIP13 (1:1000, ab128171, Abcam), rabbit anti-GAPDH (1:400; FL-335, Santa Cruz Biotechnology), mouse anti-TUBULIN (1:1000; T9026, Sigma), rabbit anti-RAD51 (1:1000; PA5-27195, Thermo Fisher Scientific) mouse anti-KU70 (1:1000; ab2172-500, Abcam), rabbit anti-Ligase IV (1:1000； ab193353, Abcam). Band intensities were assessed using Image Studio Lite (Version 5.2).
3
Cell Morphology and DNA Repair Protein Localization
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Images and cell length measurements were obtained using an Olympus IX71 microscope equipped with a personal Delta Vision system and a Photometrics CoolSnap HQ2 monochrome camera, with Metamorph software (Universal Imaging, Molecular Devices, Downingtown, PA, USA). Measurements were made from micrographs using the IMAGEJ (National Institutes of Health). All microscopy was conducted on live midlog-phase cells placed on slides, except for cultures for DAPI and aniline blue staining, which were fixed in 70% ethanol at room temperature, washed, and pelleted before resuspension in 5 μl of DAPI solution (500 μg/ml). More than 200 dividing cells per strain were measured for the cell length data. Stacks of ten z-series sections were acquired at 0.2-μm intervals. All fluorescence images are maximum two-dimensional (2D) projections of z-series and were analyzed using deconvolution software from Applied Precision. To calculate the area of the nucleus occupied by Rad52p-YFP a binary mask was created using the mean value of the nuclear background without foci as a threshold and measuring the area of this mask. To detect Rad51p indirect immunofluorescence microscopy was performed according to the protocol described in (46 (link)). The rabbit polyclonal anti-human Rad51p (PA5-27195, Thermo Fisher, Rockford, IL, USA) was diluted 1.100.
4
Western Blot Analysis of DNA Damage Response Proteins
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OVCAR4 and Ov4Carbo (4 × 105 cells/well) were seeded in six-well plates. Cells were washed with PBS and sonicated (Bioruptor Pico-RM 343 for 10 min) in ice-cold lysis buffer (150 mM NaCl, 50 mM Tris, 0.05% SDS and 1% triton), supplemented with 1% protease/phosphatase inhibitor cocktail (Roche). Samples were centrifuged (13,000×g, 4 °C, 20 mins) and protein concentration determined (Pierce BCA assay kit (Thermo Scientific)). In total, 30 μg/20 μl protein were prepared in SDS loading buffer, boiled for 5 min, separated on 4–12% Bis–Tris gels (Life Technologies) and transferred to nitrocellulose membranes (GE Healthcare) by semi-dry transfer (Trans-Blot® SD Cell, BioRad). Membranes were blocked in 5% non-fat milk/PBS for 1 h at room temperature and incubated with primary antibodies for p53 (1:1000, Sc-126 Santa Cruz, n = 4 biological repeats), RAD51 (1:1000, PA5-27195, Invitrogen, n = 3 biological repeats), GADPH (1:2000, Sc-47724 Santa Cruz), Tubulin (1:2000, ab52866 Abcam) and γH2AX (ser139 1:500, JBW301 Millipore, n = 8 biological repeats) at 4 °C overnight, followed by incubation with secondary anti-mouse HRP (1:2000, P0260 DAKO) and anti-rabbit HRP (1:1000, P0448 DAKO) for 1 h at room temperature. Proteins were visualised by enhanced chemiluminescence (GE Healthcare), imaged (Amersham Image 600, GE Healthcare) and quantified by densitometry (ImageQuant TL software package).
5
Western Blot Analysis of DNA Repair Proteins
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The mice prostate tissues or human prostate cell lines were extracted and concentrated using RIPA lysis buffer containing protease inhibitors (Sigma-Aldrich, St. Louis, MO) and BCA Protein Assay kit (Vigorous Biotechnology, Beijing, Beijing, China), respectively. Equal proteins were resolved using SDS-PAGE before being transferred onto nitrocellulose membranes (Millipore, Madison, WI). Membranes were blocked with 5% BSA in TBST buffer and incubated with following primary antibodies (LPCAT1 antibody (1:200; 66044-1-Ig; Proteintech Group, Inc., Chicago, IL); DNA-PKcs antibody (1:200; ab32566; Abcam); p-DNA-PKcs (S2056) (1:400; ab18192; Abcam), and RAD51 antibody (1:200; PA5-27195; Invitrogen™) at 4°C overnight and HRP-coupled secondary antibodies at room temperature for 1h. GAPDH was used as internal control.
6
Evaluating DNA Damage Response in OVCAR4 and Ov4Carbo Cells
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OVCAR4 and Ov4Carbo cells (8 × 104 cells/well) were seeded in duplicate on poly-l-lysine-coated coverslips and allowed to adhere overnight. Cells were then treated with chloroxine (10 μM), carboplatin (50 μM or 100 μM) or combination [(chloroxine (10 μM) + carboplatin 50 μM or 100 μM)] for 4 and 24 h. The medium was aspirated and 0.1% Triton (Sigma-Aldrich) in PBS added for 1 min prior to fixing in 3% paraformaldehyde/2% sucrose for 30 min. Cells were then blocked with 3% BSA/PBS for 1 h. Next, staining was performed with antibodies for γH2AX (1:800, Millipore JBW301) and RAD51 (1:1000, Invitrogen PA5-27195) for 40 min at 37 °C. Cells were incubated with secondary antibodies (AlexaFluor-488 and AlexaFluor-568, Invitrogen) for 30 min at 37 °C, and co-stained with DAPI (1:10,000, 1 mg/ml Sigma). Coverslips were mounted with Mowiol and images captured using a Zeiss 710 confocal microscope. Foci were counted and scored manually using ImageJ software. At least 100 nuclei were counted for each condition (using duplicate plate wells) and cells with >5 foci/nucleus were considered positive (three biological replicates were conducted).
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