Cells were processed for immunofluorescence analysis as previously described.7 (link) Slides were observed under the fluorescence microscope (Carl Zeiss Axio Imager.Z2, Jena, Germany). For microirradiation studies, cells were subjected to localized damage in the form of specific laser tracts through a UV-A laser beam. Cells were seeded in multi-chamber glass slides (Nunc, Rochester, NY, USA) and pre-sensitized with 10 μM 5-bromo-2′-deoxyuridine (Sigma) in phenol red-free medium (Invitrogen) for 24 h. Laser microirradiation was performed by Axio observer D1 microscope (Carl Zeiss), which is supplemented with PALM microbeam. Cells were exposed to 405 nm laser diode (6 mW) using PALM robo software 4.3 SP1 (Jena, Germany), focused through a × 40 objective to yield a spot size of 0.5–1 mm. The time of exposure was set in fast scanning mode with laser settings in such a range to generate a detectable laser path without causing physical damage to the cells. After microirradiation, imaging of cells was done as described above. Immunostaining for Ku70 was done as described previously.37 (link)
Phenol red free medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
Phenol red-free medium is a cell culture media formulation that does not contain the pH indicator phenol red. This allows for more accurate colorimetric measurements and detection of certain analytes in the cell culture environment.
Automatically generated - may contain errors
Lab products found in correlation
Serum replacement 2, by Merck Group (4 mentions)
Xct790, by Merck Group (3 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Ethanol, by Merck Group (2 mentions)
Tcs sp5 microscope, by Leica (2 mentions)
Fetal bovine serum (fbs), by Sartorius (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
5 bromo 2 deoxyuridine, by Merck Group (1 mentions)
Axio observer d1 microscope, by Zeiss (1 mentions)
Axio imager z2, by Zeiss (1 mentions)
Facscalibur, by BD (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Propidium iodide, by BD (1 mentions)
Vegf a, by R&D Systems (1 mentions)
Fluoview 1000 confocal microscope, by Olympus (1 mentions)
Camptothecin, by Merck Group (1 mentions)
Faxitron cellrad, by Hologic (1 mentions)
35 protocols using phenol red free medium
1
Laser Microirradiation Imaging Protocol
2
Quantifying Autophagy via AO Staining
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Autophagy quantification by AO staining using flow cytometry was performed as previously described [50 (link)]. Briefly, cells were stained with 1μM AO for 15 minutes at 37°C, trypsinized, washed, and re-suspended in phenol red-free medium (Invitrogen) supplemented with 10% fetal bovine serum (HyClone). After filtration, single cell solution was detected by flow cytometry (BD FACS Calibur) with 488nm excitation light. Autophagy was quantified as a ratio between geomean fluorescence intensity of red vs. green fluorescence (FL3/FL1).
3
Cell Cycle Analysis of Drug Treatments
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Cells were seeded at a density of 1×105 cells/well into 6-well plates and cultured until 80% confluence. Then, cells were transferred to phenol red-free medium (Invitrogen, Carlsbad, CA, USA) containing 1% serum-replacement-2 ( Sigma-Aldrich, St. Louis, MO, USA) for 24 h and were treated with TAM, RAL, XCT, T+X, R+X, or no drugs as a control for 24 h. Cells were fixed and stained with propidium iodide (PI; 100 μg/ml) (BD Biosciences, Franklin Lakes, NJ, USA) and then analyzed by BD FACSCantoⅡ™ flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) for cell cycle analysis. All experiments were performed in triplicate.
4
Isolation and Culture of Primary Mouse Heart Endothelial Cells
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Primary heart endothelial cells were isolated from ketamine/xylazine-anesthetized 2-to-3–month-old mice, as previously described [25 (link)]. Briefly, the heart was Collagenase A-digested and cells were sorted by anti-Mouse CD31, immobilized on Sheep anti-Rat IgG Dynabeads, and cultured on 0.1% gelatin pre-coated plates in growth medium [DMEM (Gibco Lab., Gaithersburg, MD, USA), 0.01% heparin, 10% Bovine Endothelial Cell Growth Supplement-ECGS (Cell Applications, Inc-San Diego, CA, USA), 20% FBS, 1% Penicillin–Streptomycin] for ~3 weeks until they reach ~80% confluency. Cells were then seeded at a density of 3 × 105 cells/well in 6-well plates and allowed to grow for 24 h before being serum-starved for 2 h in phenol red-free medium (Invitrogen, Waltham, MA, USA) supplemented with 25 mM HEPES and 0.1% BSA before being incubated in the presence or absence of 40 ng/mL VEGF-A (R&D systems, Minneapolis, MN, USA) for 5 or 20 min for Western blot and media analysis, respectively.
5
Laser-Induced DNA Damage Assay
6
Endometrial Cancer Cell Line Treatment
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Human RL952, HEC-1A, and HEC-1B endometrial adenocarcinoma cells were obtained from the Shanghai Cell Biological Research Institute (Shanghai, China), and ECC-1 cells were acquired from the American Type Culture Collection (ATCC, Manassas, VA, USA). RL952 and ECC-1 cells are ERα+, while HEC-1A and HEC-1B cells are ERα-. RL952 and ECC-1 cells were thawed and cultured in DMEM/F12 medium with 0.005 mg/ml insulin, 1% antibiotic–antimycotic solution, and 10% fetal bovine serum (FBS) or in RPMI-1640 medium supplemented with 10% FBS. HEC-1A and HEC-1B cells were cultured in high-glucose DMEM supplemented with 10% FBS at 37°C in 5% CO2. XCT790, TAM, and RAL were purchased from Sigma (St. Louis, MO, USA) and were dissolved in dimethyl sulfoxide (DMSO) at 25°C. Aliquots of stock solutions at 1 mM were stored at −20°C. Cells were transferred to phenol red-free medium (Invitrogen, Carlsbad, CA, USA) containing 1% serum-replacement-2 ( Sigma-Aldrich, St. Louis, MO, USA) for 24 h. Then, cells were treated for various lengths of time with TAM (10 μM), RAL (10 μM), XCT790 (XCT; 10 μM), TAM + XCT790 (T+X; 10 μM), RAL + XCT790 (R+X; 10 μM), or no drugs as a blank control.
7
Hormone-Depleted MCF-7 Estrogen Response
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MCF-7, a breast cancer cell line, was cultured in DMEM/F12 (Gibco) supplemented with 10% FBS and 1% penicillin-streptomycin and maintained at 37 °C, 5% CO2 humidified incubator. Before 4C-seq assays, MCF-7 cells were grown in hormone-free media: they were washed with PBS twice to remove any residual FBS or growth factors and incubated in phenol red-free medium (Invitrogen/Gibco) supplemented with 10% charcoal-dextran stripped FBS (Hyclone) and 1% pencillin-streptomycin for a minimum of 72 h. Hormone-depleted MCF-7 cells were then treated with estrogen (Sigma) to a final concentration of 100 nM for 45 min before 4C-seq assay. The control cells were treated with an equal volume and concentration of vehicle, ethanol (Sigma), for 45 min.
8
FRAP analysis of nuclear dynamics
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HeLa cells were plated on 35-mm high glass-bottom Ibidi μ-dishes, in phenol red-free medium (Invitrogen). FRAP experiments were conducted on a Leica TCS SP5 microscope equipped with a 63 × 1.4 numerical aperture oil-immersion lens. During experiments, cells were maintained at 37 °C and 5% CO2. A defined circular region of 2 μm diameter (Region of Interest 1 — ROI1) was placed to the nuclear midpoint of cells. GFP was excited using the 488 nm Argon laser line. Fifty pre-bleach images were acquired with 2% of the 488 nm line at 60% Argon laser intensity, followed by double bleach pulses on ROI1 of 0.066 s using the 476 nm and 488 nm laser lines, combined at maximum power. In this manner, at least 60% of the fluorescence in ROI1 was successfully bleached. Following bleaching, 300 images were recorded at 0.066 s intervals. Mean fluorescence intensities of the ROI1, the whole nucleus (ROI2) and an area outside the nucleus for background correction (ROI3) were quantified and exported as comma-separated values. Quantitative analysis of the experimental recovery curves was performed using easyFRAP [29] (link) and model-based analysis was performed using the parameter inference method described previously [30] (link).
9
Local UV Laser Micro-irradiation of HeLa Cells
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HeLa cells were seeded on 15 mm diameter glass-bottom plates (NEST, Wuxi, China) and presensitized with 10 μM Brdu (Roche, Basel, Switzerland) in phenol red-free medium (Invitrogen, USA) for 24 h at 37°C. Local laser micro-irradiation was performed by a micropoint system (Andor, Belfast, UK) with a 365 nm pulsed nitrogen UV laser (16 Hz pulse, 55% laser output). After irradiation, cells were immunostained as previously described (27 (link)) with anti-γH2AX antibody (#9718, Cell Signaling Technology). The images were captured using the Axion Vision Rel.4.6 computerized image analysis system (Carl Zeiss, Jena, Germany).
10
FRAP Analysis of Intracellular Dynamics
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Cells (HeLa or MCF7) were plated on 35-mm glass-bottom dishes in phenol-red–free medium (Invitrogen). FRAP experiments were conducted as previously described (Xouri et al, 2007b (link); Giakoumakis et al, 2017 (link)). A Leica TCS SP5 microscope equipped with a 63x magnification 1.4 NA oil-immersion lens and FRAP booster were used. During FRAP, cells were maintained at 37°C and 5% CO2. A defined circular region of interest with 2 μm diameter (ROI1) was placed in the nucleus and at the recruitment sites. GFP was excited using a 488-nm argon laser line. 50 pre-bleach images were acquired with 3% of the 488-nm line at 60% argon laser intensity, followed by a single bleach pulse on ROI1 using 476-nm and 488-nm laser lines combined at maximum power. In this manner, at least 60% of the fluorescence in ROI1 was bleached successfully. Next, 400 post-bleach images with a 0.052 s interval were recorded. Mean intensities of ROI1, the whole nucleus (ROI2) and an area outside of the nucleus (ROI3) were quantified and exported as .csv format files. Data analysis was performed using easyFRAP (Rapsomaniki et al, 2012 (link); Giakoumakis et al, 2017 (link); Koulouras et al, 2018 (link)).
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