Seventy-two hours after treatment with Doxycycline to induce shRNA for AR, C4-2 cells were exposed to IR and washed twice with PBS and collected by trypsinisation. Approximately 5 × 103 cells in 10 μl of PBS (−) were mixed with 90 μl of LMAgarose (Trevigen), placed on GelBond Film (Lonza), covered with a 22 mm cover slide (VWR International) and left at 4 °C for 1 h. On removal of the cover slide, the cells were lysed with lysis solution (Trevigen) at 4 °C for 1 h. Following a wash with TBE (90 mM Tris borate (pH 8.3) and 2 mM EDTA), the samples were subjected to electrophoresis at 35 V, for 7 min in TBE. After washing with TBE, samples were fixed with 70% ethanol for 5 min at room temperature and dried overnight. The nuclei were stained with SYBR Green I (Invitrogen) in 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA for 5 min at 4 °C. Images were taken with an IX71 fluorescent microscope (Olympus) with Cell∧F software (Olympus). Tail moments were measured using CometScore software (TriTek). The means of tail moment of at least 30–50 cells were measured per condition. Efficiency of DSB repair was determined as the tail moment ratio between the time points and the undamaged control cells obtained immediately after treatment.
Gelbond film
Manufactured by Lonza
Sourced in United States
GelBond film is a transparent polyester film used as a support for gel electrophoresis. It provides a stable and inert surface for casting gels.
Automatically generated - may contain errors
Lab products found in correlation
Cell f software, by Olympus (3 mentions)
Sybr green 1, by Thermo Fisher Scientific (3 mentions)
Ls positive selection column, by Miltenyi Biotec (3 mentions)
Countbright absolute counting beads, by Thermo Fisher Scientific (3 mentions)
Sybr gold nucleic acid gel stain, by Thermo Fisher Scientific (3 mentions)
Quadromacs separator, by Miltenyi Biotec (3 mentions)
Lympholyte mammal cell separation reagent, by Cedarlane (3 mentions)
Anti pe microbeads, by Miltenyi Biotec (3 mentions)
Nusieve gtg agarose, by Lonza (3 mentions)
Ix71 fluorescent microscope, by Olympus (2 mentions)
Lmagarose, by Bio-Techne (2 mentions)
22 mm cover slide, by Avantor (2 mentions)
Lysis solution, by Bio-Techne (2 mentions)
Heat inactivated foetal bovine serum, by GE Healthcare (2 mentions)
Dulbecco phosphate buffered saline (dpbs), by Lonza (1 mentions)
Ultrapure agarose, by Thermo Fisher Scientific (1 mentions)
Ultrapure low melting point agarose, by Thermo Fisher Scientific (1 mentions)
Low melting agarose, by Bio-Techne (1 mentions)
Phosphate buffered saline (pbs), by Lonza (1 mentions)
Agarose, by Bio-Rad (1 mentions)
18 protocols using gelbond film
1
Comet Assay for DNA Double-Strand Break Repair
2
Agarose Microwell 3D Spheroid Culture
3
Comet Assay for DNA Damage Quantification
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We used a protocol for quantifying DNA damage of cells by the single cell comet electrophoresis method in multiple mini-gels previously described in (50 (link)). Brain regions from 20-month-old mice were dissected and homogenized in 1X phosphate buffered saline (PBS) on ice. The homogenate was passed through a 0.45-μm filter to catch any remaining tissue fragments. The extracts were then spun down at 800 x g for 10 min at 4°C. The supernatant was removed and the nuclei pellet weighed and re-diluted in the appropriate ratio of 1X PBS. The nuclei were added to melted agarose at a ratio of 4:1. Approximately 4 μl of agarose cell suspension was dotted onto the hydrophilic side of the GelBond film (Lonza), repeated as necessary for each sample using a multi-channel pipette. The membrane was allowed to cool and the cells lyzed overnight at 4°C, using standard neutral comet protocol (50 (link)). We ran the electrophoresis at 25 V for 30 min at 4°C and stained with 1x SYBR Gold (Life Technologies). Comets were imaged using a fluorescent microscope and comet analysis software. All four mice were run together on a single film for each section, reducing variability. At least 50 comets were taken and the assay was repeated with different ages with similar trends always observed in the data. Statistical analysis was conducted on the data using one-way ANOVA.
4
Fabrication of Comet Chip Agarose Gels
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For the Comet Chip assay, 96-well agarose gels were prepared by using molten 1% normal melting point (NMP) agarose (ThermoFisher, Waltham, MA, USA) in PBS. The molten agarose was poured onto the hydrophilic side of a Gel Bond® film (Lonza, Walkersville, MD, USA), which was placed up in a rectangular petri dish lid and evenly spread across the lid. The polydimethylsiloxane (PDMS) stamp, with an array of micropegs, was gently placed on top of the gel. The gel was allowed to solidify for 15 min. The PDMS was removed, and 10 mL PBS was added to the dish. The micropore formation was confirmed by observing the gel on an inverted microscope with 10× magnification. The excess gel from the lid was removed, and the gel attached to the Bond® film was submerged in PBS and stored before use at 4 °C for no longer than a week.
5
Silicone Elastomer Hydrogel Fabrication
6
Quantifying DNA Damage by Comet Assay
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U2OS cells were treated with G9a inhibitor (UNC0638, 1 µM) or ATM inhibitor (KU55933 [16] (link), 10 µM) for 24 h, while HCT116 p53 WT/KO cells were treated for 4 days. For siRNA-transfected cells, assays were performed 72 h after transfection. Neutral comet assay were performed with the damaging agent phleomycin, as previously reported [17] (link). Briefly, an appropriate number of cells were mixed with low-melting Agarose (Trevigen) and bound on GelBond film (Lonza). Samples were lysed and electrophoresed at 35 V for 7 min. The samples were fixed, dried and stained with SYBR Green I (Invitrogen). Images were taken with an IX71 fluorescent microscope (Olympus) using Cell^F software (Olympus). Tail moments were quantified using CometScore software (TriTek). Means of tail moments of at least 50 cells were measured per condition.
7
Conidiation and Appressorium Formation Assay
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For the conidiation assays, we selected 5 mm diameter mycelial plugs from the edge of a 3-days-old colony that had been placed onto PDA medium and grown at 25°C for 7 days. Conidial suspensions from each strain were selected from the PDA plates after applying 5 mL of sterile water. Conidial suspensions were placed at 25°C for 12 h on a GelBond membrane to allow germination and growth. The suspensions were monitored, and the colony-forming efficiency was assessed under the microscope.
Equal numbers of spores were placed on the hydrophobic surface of the GelBond film (Lonza) at 28°C for 12 h to examine appressoria formation. The appressoria from germinated spores were counted. At least 200 conidia were surveyed in experiments. The rates of conidiation and appressorium formation assays were performed in triplicate, in three independent experiments.
Equal numbers of spores were placed on the hydrophobic surface of the GelBond film (Lonza) at 28°C for 12 h to examine appressoria formation. The appressoria from germinated spores were counted. At least 200 conidia were surveyed in experiments. The rates of conidiation and appressorium formation assays were performed in triplicate, in three independent experiments.
8
Double-Strand Break Repair Efficiency
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Seventy-two hours after 40 nM siRNA transfection, the cells were incubated with 40 μg/ml phleomycin for 2 hours or mock incubated. Following phleomycin treatment, cells were washed twice with PBS and cultured for an additional 2 hours. The cells were subsequently washed twice with PBS (−) and collected by trypsinization. Approximately 5 × 103 cells in 10 ml of PBS (−) were mixed with 90 μl of LMAgarose (Trevigen), placed on GelBond Film (Lonza), covered with a 22 mm cover slide (VWR International) and left at 4 °C for 1 hour. Upon removal of the cover slide, the cells were lysed with Lysis Solution (Trevigen) at 4 °C for 1 hour. Following a wash with TBE [90 mM Tris-Borate (pH 8.3) and 2 mM EDTA], the samples were subjected to electrophoresis at 35 V, for 7 min in TBE. After washing with TBE, samples were fixed with 70% ethanol for 5 min at room temperature and dried overnight. The nuclei were stained with SYBR Green I (Invitrogen) in 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA for 5 min at 4 °C. Images were taken with a fluorescent microscope IX71 (Olympus) with Cell^F software (Olympus). Tail moments were measured by using CometScore software (TriTek). The means of tail moment of at least 50 cells were measured. Efficiency of DSB repair was determined as the tail moment ratio between 2 hours after phleomycin removal and immediately after treatment.
9
CometChip Assay for Genotoxicity Evaluation
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CometChip assay was performed as described (Wood et al., 2010 (link); Ge et al., 2012 , 2014 (link), 2015 (link); Sykora et al., 2018 (link); Weingeist et al., 2013 (link)). A homemade PDMS stamp with an array of micropillars was obtained from Professor Bevin Engelward (Department of Biomedical Engineering, Massachusetts Institute of Technology, USA). Briefly, 1% w/v agarose (BioRad, USA) was dissolved in phosphate-buffered saline (PBS) (Lonza, USA) and applied to the hydrophilic side of GelBond® film (Lonza, USA). The PDMS stamp was pressed on top of the molten agarose gel to generate arrays of microwells with around 40-50 μm in both diameter and depth, 240 μm space. The agarose gel was allowed to solidify for 15 min before the stamp was removed. A bottomless 96-well plate was applied on top of the agarose gel chip and secured by clips to form 96 macrowells, with an array of ~300 microwells in each.
10
Appressoria Formation in Magnaporthe oryzae
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