Cells were cultured under normoxia (21% O2) or hypoxia (1% O2) in a tri-gas chamber (Heracell 150i, Thermo Fisher Scientific, Waltham, MA, USA; or 9000E; Wakenyaku, Kyoto, Japan). For hypoxic conditions, cells were grown in hypoxia for 20–24 h before irradiation. During irradiation, Lab-TeksTM (Dutscher, Bruxelles, Belgium) were covered with an airtight plastic film to limit oxygen exchanges and were immediately replaced after exposure in a hypoxic incubator for kinetic studies (30 min to 24 h).
Lab tek
Manufactured by Dutscher
Sourced in Belgium, France
Lab-TeksTM is a line of lab equipment designed for various scientific applications. The product details and core functions are provided in a factual and unbiased manner without interpretation or extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Heracell 150i, by Thermo Fisher Scientific (3 mentions)
Mouse anti phospho histone h2a x ser139, by Merck Group (1 mentions)
Rabbit anti phospho dna pkcs s2056, by Abcam (1 mentions)
Rabbit anti 53bp1, by Novus Biologicals (1 mentions)
Mouse anti hif1α, by BD (1 mentions)
Rabbit anti rad51, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Fluoromount, by Merck Group (1 mentions)
Mitotracker green dye, by Thermo Fisher Scientific (1 mentions)
4 protocols using lab tek
1
Hypoxia and Radiation Exposure
2
Radiation-Induced DNA Damage Response
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Cells were seeded the day before experiments on Lab-TeksTM (Dutscher) at a density of 3.105 cells per slide and incubated at 37 °C under normoxia or hypoxia before irradiation. After radiation exposure (30 min to 24 h), cells were fixed for 15 min in 4% PFA, permeabilized, and labeled according to recommendations of the manufacturer for each primary antibody, i.e., 1 h at room temperature (1:1000, mouse anti-phospho-histone H2AX-Ser139 (Merck, Kenilworth, NJ, USA); 1:750 rabbit anti-Rad51 (Abcam, Cambridge, GB); 1:250, rabbit anti-53BP1 (NovusBio, Littleton, CO, USA); 1:250, rabbit anti-phospho-ATM-S1981 (Abcam); 1:50 mouse anti-HIF-1α (BD Transduction, San-José, CA, USA), and 1:500, rabbit anti-phospho-DNA-PKcs-S2056 (Abcam)) and secondary antibodies, i.e., for 1 h in the dark at room temperature (1:500, anti-IgG rabbit Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA); 1:250, anti-IgG mouse Alexa Fluor 555 (Abcam)). Nuclei were stained for 15 min with 1 µg/mL DAPI (Sigma, Kanagawa, Japan), then slides were mounted using Fluoromount (Merck) and stored at room temperature in the dark until analysis [38 (link)] (n = 2).
3
TUNEL Assay for Apoptosis in Primary Neurons
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For TUNEL studies primary neuronal cells obtained from Prnp+/+ and Prnp−/− animals grown on Lab-Tek (Dutscher) were treated for 1 h with 1, 2 or 3-mM MMS, washed with culture medium and returned for 6 h in the incubator at 37°C. They were then fixed for 10 min in 4% paraformaldehyde and permeabilized in PBS containing 0.1% Triton X-100 for 10 min. After a wash in PBS, cells were incubated in the dark for 1 h at 37°C, with the enzyme and the label solution provided by the ‘In Situ Cell Death Detection Kit, Fluorescein’ (Roche Diagnostic). Nuclear staining was achieved by incubation with DAPI. Slides were mounted under Fluoromount (Southern Biotechnologies Associates). Apoptosis was measured by counting TUNEL positive cells visualized by fluorescence microscopy. For each point a minimum of 1000 cells were analyzed.
4
Mitochondrial Doxorubicin Uptake Visualization
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Cells were grown in high-glucose DMEM on a 4-well LAB-TEK (Dutscher, Mérignac, France). The mitochondrial network was stained with the Mitotracker Green dye (Molecular Probes, Eugene, OR, USA), used at 100 nM for 30 min and 5 μM of DXR at 37 °C for 10 min. The cells were visualized by fluorescence microscopy. The fluorescence intensity was measured in the green (ex 490 nm/em 516 nm) and the red (ex 470 nm/em 585 nm) by using appropriate filters. For these observations, a Zeiss microscope (AxioVision, Carl Zeiss, Oberkochen, Germany) was used, with a 63× oil objective. DXR accumulation in the mitochondrial compartment was estimated with Carl Zeiss software. Pearson’s correlation coefficients were determined.
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