In order to determine the degree of DNA damage induced by the treatments, DNA double-strand breaks were visualized by γH2AX immunostaining. For this, 1 × 105 cells/well were seeded onto coverslips in 24-well plates. On the following day, the samples were treated for 3 h with 20 µM of estrone-TSC, estradiol-TSC, complexes Cu(II)-estrone-TSC and Cu(II)-estradiol-TSC or with equivalent amounts of DMSO or CuCl2 solution. Then, cells were fixed with 4% formaldehyde (Molar Chemicals, Halásztelek, Hungary), permeabilized with 0.3% Triton-X-100 (Calbiochem, Merck Millipore, Darmstadt, Germany) and were blocked using 5% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA). Then, the samples were incubated with anti-γH2AX antibody (Thermo Fisher Scientific, Waltham, MA, USA) in 1:300 dilution followed by Alexa 488 fluorophore-conjugated goat anti-mouse secondary antibody (Abcam, Cambridge, UK) in 1:600 dilution. The fluorescence intensity of the stained samples was detected by an Olympus FV10i confocal microscope. For the positive control samples, DNA double-strand breaks were induced by exposing cells to a 2 Gy dose of ionizing radiation for 1 min using an X-ray system (RS320, Xstrahl Limited, Xstrahl Surrey, UK) at the Extreme Light Infrastructure Attosecond Light Pulse Source (ELI-ALPS) Research Institute.
Rs320
Manufactured by Xstrahl
Sourced in United Kingdom
The RS320 is a laboratory x-ray source designed for research and educational applications. It generates x-rays through the acceleration of electrons and their subsequent collision with a metal target. The core function of the RS320 is to produce x-rays for experimental and analytical purposes.
Automatically generated - may contain errors
Lab products found in correlation
Anti γh2ax antibody, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Fv10i confocal microscope, by Olympus (1 mentions)
Nocodazole, by Merck Group (1 mentions)
Plasmocin, by InvivoGen (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Nightowl lb 983, by Berthold Technologies (1 mentions)
Celltracker cm dii dye, by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Cell proliferation dye efluor 450, by Thermo Fisher Scientific (1 mentions)
Phytohemagglutinin p pha p, by Merck Group (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
10 protocols using rs320
1
Quantifying DNA Damage via γH2AX Immunostaining
2
Ionizing Radiation Exposure in Mice
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Mice were anesthetized with 2% isoflurane and irradiated in 0.8% isoflurane with either a single dose of 0 Gy (sham control treatment) or with 10 Gy ±5% in 5 mm tissue depth (~1.53 Gy/min, 300 kV, filter: 0.5 mm Cu, 10 mA, focus distance: 60 cm) using a collimated beam with a XStrahl RS 320 cabinet irradiator (XStrahl Limited, Camberly, Surrey, Great Britain) (Panic et al., 2017 (link)). Mice were humanely sacrificed at indicated time points with CO2 inhalation and transcardial perfusion, and tumor tissue was isolated for respective downstream analysis.
3
X-ray Irradiation of Cell Cultures
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Irradiation was performed with an X-ray system, (RS320, Xstrahl Limited) at the Extreme Light Infrastructure Attosecond Light Pulse Source (ELI-ALPS) Research Institute. 250 keV energy X-ray beam was used to irradiate the cells seeded into 6- and 96-well plates at a dose rate of 3.65 Gy/min. The plates were placed into a special polymethyl methacrylate (PMMA) slab phantom [28 (link)], with the isocenter positioned at the geometrical centers of the plates. The delivered doses (0, 2, 5, and 8 Gy) were verified by performing dosimetry measurements in a regular manner using a calibrated ionization chamber (Farmer type ionization chamber–PTW TM30013), film dosimetry (Gafchromic EBT self-developing dosimetry film) and modified FBX type dosimeters prepared in our laboratory [28 (link)]. The combined standard uncertainty of the measurements was 2.2%. For each irradiated plate, control measurements were performed using Gafchromic EBT3 films.
4
X-ray irradiation of spheroid cultures
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An X-ray irradiator RS320 (Xstrahl Ltd., Surry, UK) was used to irradiate the spheroid cultures at 300 kV, 10 mA, and a dose rate of 0.9 Gy/min. Irradiation was performed in 100 µL medium with or without carvedilol in multi-wells with 5 or 10 Gy within one hour.
5
Cell Synchronization and Perturbation Assay
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For synchronization, HeLa cells were blocked in mitosis with 300 nM nocodazole (Sigma) for 22 h. Cells were infected 4 h after release in nocodazole free growth medium. Etoposide treatment of cells was performed for 24 h using a concentration of 40 µM. Cells were exposed to 10 Gray X-ray irradiation using an Xstrahl RS320 irradiator. Cycloheximide treatment was performed using a concentration of 100 µM.
MG132 treatment was performed for 5 h prior to infection and was used at a concentration of 10 µM. Pepstatin-A-methylester (PME) was used for 1 h prior to infection at a concentration of 200 µM. 20 mM EGTA was added to cells upon infection.
Cytochalasin D treatments were performed for 15 minutes prior to infection at a concentration of 5 µg/ml.
MG132 treatment was performed for 5 h prior to infection and was used at a concentration of 10 µM. Pepstatin-A-methylester (PME) was used for 1 h prior to infection at a concentration of 200 µM. 20 mM EGTA was added to cells upon infection.
Cytochalasin D treatments were performed for 15 minutes prior to infection at a concentration of 5 µg/ml.
6
Spheroid Radiation and Electrochemotherapy
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The spheroid cultures were irradiated and/or treated with electrochemotherapy (ECT). In terms of combination therapy, the spheroid cultures were first irradiated and additionally treated with ECT within one hour after radiation. As controls, the spheroid cultures remained untreated.
Radiation: spheroid cultures were irradiated in a 100 µL medium in multi-wells with a dose of 5 or 20 Gy by using a 60Co source X-ray irradiator (RS320, Xstrahl Ltd., Surrey, UK) at 300 kV, 10 mA and a dose rate of 0.9 Gy/min.
ECT: the spheroids were treated with a 100 µL medium containing 1.0 µg/mL or 2.5 µg/mL bleomycin and electroporated by using two parallel aluminum electrodes 4 mm apart. Eight pulses (100 µs pulse duration, 5 Hz repetition frequency) of a 1000 V/cm or 750 V/cm pulse strength were applied by a voltage pulse generator (Genedrive, IGEA S.p.A., Carpi, Italy). As controls, further samples were treated with bleomycin or electroporation only. After 24 h of treatment, the spheroids were washed with the medium and incubated in a fresh medium for the indicated period of time.
Radiation: spheroid cultures were irradiated in a 100 µL medium in multi-wells with a dose of 5 or 20 Gy by using a 60Co source X-ray irradiator (RS320, Xstrahl Ltd., Surrey, UK) at 300 kV, 10 mA and a dose rate of 0.9 Gy/min.
ECT: the spheroids were treated with a 100 µL medium containing 1.0 µg/mL or 2.5 µg/mL bleomycin and electroporated by using two parallel aluminum electrodes 4 mm apart. Eight pulses (100 µs pulse duration, 5 Hz repetition frequency) of a 1000 V/cm or 750 V/cm pulse strength were applied by a voltage pulse generator (Genedrive, IGEA S.p.A., Carpi, Italy). As controls, further samples were treated with bleomycin or electroporation only. After 24 h of treatment, the spheroids were washed with the medium and incubated in a fresh medium for the indicated period of time.
7
Cell Culture and X-ray Irradiation of NSCLC
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The human non-small cell lung carcinoma NSCLC cell lines A549 and H460 were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). The H460 cell line was maintained in RPMI-1640 (Invitrogen, Paisley, UK) supplemented with 10% FBS plus antibiotics. The A549 cell line was maintained in MEM (Invitrogen) supplemented with 15% FBS plus 1% non-essential amino acids and antibiotics. NSCLC cell lines were propagated in medium supplemented with 5 µg/ml plasmocin (InvivoGen, Toulouse, France) as a mycoplasma prophylaxis and were cultivated in an atmosphere consisting of 5% CO2 and 95% air at 37 °C. Both cell lines were usually re-thawed after 3 month in culture. Cells were irradiated using an X-ray machine RS320 (Xstrahl Ltd, Surrey, UK) at 300 kV, 10 mA, dose rate 0.9 Gy/min.
8
Generating Xenograft Tumors for Radiation Therapy
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Mouse xenograft tumours were generated by subcutaneous injection of 0.5 × 106 PC3 cells (+/−CAV1) either alone or mixed with 0.5 × 106 WMPY-1 cells (+/−TRIAP1) onto the hind limb of male NMRI nude mice (total volume 50 µL) as previously described [11 (link),21 (link)]. Animals of each experimental group received a single subcutaneous injection. For radiation therapy mice were anesthetized (2% isoflurane) and tumours were exposed to a single dose of 10 Gy ± 5% in 5 mm tissue depth (~1.53 Gy/min, 300 kV, filter: 0.5 mm Cu, 10 mA, focus distance: 60 cm) using a collimated beam with an XStrahl RS 320 cabinet irradiator (XStrahl Limited, Camberly, Surrey, Great Britain). Mouse experiments were carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the German Government and they were approved by the Committee on the Ethics of Animal Experiments of the responsible authorities [Landesamt für Natur, Umwelt und Verbraucherschutz (LANUV), Regierungspräsidium Düsseldorf Az.8.87-50.10.37.09.187; Az.8.87-51.04.20.09.390; Az.84-02.04.2015.A586].
9
Tracking Senescent Cell Engraftment
10
Suppression of T Cell Proliferation
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CD3+ cells (CD3+CD25low/intCD127+, Figure S1 ) were sorted as responder cells and stained with 20 μM Cell Proliferation Dye eFluor450 following manufacturer's protocol (CPD; eBioscience Inc., San Diego, CA, USA). Autologous Tregs (CD4+CD45RO+CD25hiCD127–, Figure S1 ) or cultured memory CCR6+ Th cells (see Cell culture) were used as putative suppressors and stained with 5 μM carboxyfluorescein succinimidyl ester (CFSE; LifeTechnologies, Eugene, OR, USA) as described by others (33 (link)). In 96-well plates 2.5 × 104 responder cells were co-cultured with 2.5 × 104 suppressor cells per well, under stimulation of 2.5 × 104 irradiated autologous PBMC (40 Gy, RS320, X-strahl, Surrey, UK) and 10 μg/ml phytohemagglutinin P (PHA-P, Sigma-Aldrich). Where indicated, 20% cell-free synovial fluid diluted in culture medium was added. Proliferation was measured on the FACSCantoII Flow Cytometer (BD Biosciences, San Diego, CA, USA) after 6 days.
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