Iron tri(acetylacetonate) (2 mmol), 1,2-tetradecanediol (10 mmol), oleic acid (6 mmol), oleylamine (6 mmol), dibenzyl ether, citric acid, diethyl ether, 2-methoxyethylamine, N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-Hydroxysuccinimide (NHS), thioglycolic acid, hydroxylamine, and phenanthroline were all purchased from Sigma-Aldrich (St. Louis, Missouri). 1,2-dichlorobenzene and N,N’-dimethylformamide were from Acros Organics (Morris Plains, New Jersey). NH2-PEG (550 Da, 2000 Da, 5000 Da, and 10000 Da) were all obtained from Laysan Bio, Inc. (Arab, Alabama). Slide-A-Lyzer G2 dialysis cassettes, chambered slides, bovine calf serum (BCS) were acquired from Thermo Scientific (Rockford, Illinois). Dulbecco’s Modified Eagle’s Medium (DMEM), Dulbecco’s Phosphate Buffer Saline (DPBS), penicillin, and streptomycin were purchased from Gibco Life Technologies, Inc. (NY, USA).
Chambered slides
Manufactured by Thermo Fisher Scientific
Sourced in United States
Chambered slides are a type of laboratory equipment used for various cell culture and microscopy applications. They provide a controlled environment for the cultivation and observation of cells. Chambered slides consist of a glass or plastic substrate with one or more enclosed compartments, allowing for the independent culture and analysis of multiple samples simultaneously.
Automatically generated - may contain errors
Lab products found in correlation
Thioglycolic acid, by Merck Group (1 mentions)
Phenanthroline, by Merck Group (1 mentions)
Hydroxylamine, by Merck Group (1 mentions)
1 2 tetradecanediol, by Merck Group (1 mentions)
Bovine calf serum bcs, by Thermo Fisher Scientific (1 mentions)
Slide a lyzer g2 dialysis cassette, by Thermo Fisher Scientific (1 mentions)
N n dimethylformamide (dmf), by Thermo Fisher Scientific (1 mentions)
Iron tri acetylacetonate, by Merck Group (1 mentions)
2 methoxyethylamine, by Merck Group (1 mentions)
1 2 dichlorobenzene, by Thermo Fisher Scientific (1 mentions)
Oleic acid, by Merck Group (1 mentions)
Oleylamine, by Merck Group (1 mentions)
Citric acid, by Merck Group (1 mentions)
Diethyl ether, by Merck Group (1 mentions)
Dibenzyl ether, by Merck Group (1 mentions)
Alexa fluor secondary antibodies, by Abcam (1 mentions)
Cytation c10, by Agilent Technologies (1 mentions)
Anti fade mounting media, by Vector Laboratories (1 mentions)
Nuclear fast red, by Vector Laboratories (1 mentions)
Eclipse 50i, by Nikon (1 mentions)
6 protocols using chambered slides
1
Synthesis and Characterization of Iron Nanoparticles
2
Immunofluorescence Staining of NSCs and Astrocytes
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Cells were plated on 8 chambered slides (ThermoFisher) and treated with MNPLs for 24 h after 7 days of differentiation. Cells were washed 3× with PBS before being fixed with 4% formaldehyde in PBS pH 7.4 for 15 min and then permeabilized with 0.5% Triton X-100 in 1× PBS for 15 min. Cells were then blocked with 10% fetal bovine serum (FBS) for 2 h at room temperature. NSCs and astrocytes were incubated overnight at 4 °C with primary antibodies α-Nestin (1:200; Novus Biologicals, Toronto, ON, Canada) and α-GFAP (1:500; Cell Signaling, Danvers, MA, USA), respectively. Cells were washed with PBS and then incubated with Alexa Fluor™ secondary antibodies (1:500, GαChicken (Nestin) Alexa 594 Abcam, Boston, MA, USA) (1:500, GαRabbit (GFAP) Alexa 568 ThermoFisher) diluted in 1% FBS, 0.1% Triton X-100 for 2 h at room temperature. Cells were washed 3× with PBS and mounted in ProLong Diamond Antifade mounting media containing DAPI for staining nuclei (ThermoFisher). Cells were visualized by microscopy at 40× using the Cytation C10 (Agilent Technologies, Santa Clara, CA, USA) microscope.
3
Cytochrome C Immunostaining Protocol
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1 × 106 cells were seeded in the chambered slides (ThermoFisher, Carlsbad, CA, USA) and left for overnight attachment, followed by the treatment of CBD or Cisplatin or Vehicle control for 6h. Then slides were fixed with 4% formaldehyde and the blocked with 5% normal serum 0.3% tritonx100 for 1 h. After blocking slides were incubated with primary Cyt-c (1:100) (CST, Danvers, MA, USA) primary antibody overnight at 4 degrees. After 3 gentle washes with PBS slides were incubated with HRP conjugated secondary antibody (1:500) for 1h in dark followed by 3 washes with PBS and were mounted with antifade mounting media (Vectashield, Burlingame, CA, USA).
4
Prussian Blue Staining for SPION Uptake
5
Radiation Exposure and Radioprotectant Evaluation
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Mice were irradiated with a single, total-body radiation dose (3 Gy) in a Shepherd Mark I model 25 137Cs irradiator (J. L. Shepherd & Associates, San Fernando, CA). The average dose rate was 0.98 Gy/min and was corrected for decay. The animals were placed in a well-ventilated rotating chamber composed of T-6061 aluminum with a gold-anodized coating. The chamber was divided into eight equal “pie slice” compartments with a well-ventilated Plexiglas lid (J.L. Shepherd & Associates). Animals were injected subcutaneously into the loose skin over the neck with a single dose of GT3 or vehicle (5% Tween-80 in saline) 24 h before irradiation. All radiation experiments were performed in the morning to minimize diurnal effects. HUVECs were irradiated in T25 tissue-culture flasks (Corning, NY) or chambered slides (Nunc Lab-Tek, St. Louis, MO). HUVECs were treated with 0–5 μM of GT3 or vehicle (DMSO) prior to irradiation. Radiation doses used for the various studies were carefully selected based on experimental endpoints and knowledge regarding cellular radio-sensitivity.
6
High-Cholesterol Diet Effects on Mouse Hepatocytes
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CD1 male mice (8 to 10 weeks old) were maintained in pathogen-free housing and cared for in accordance with the Universidad Autónoma Metropolitana and NIH Guide for the Care and Use of Laboratory Animals.
Twenty mice were randomly divided in two groups; in HC group 10 animals were fed with a high-cholesterol diet (HC, 2% cholesterol and 0.5% sodium cholate) for two days, as reported by Marí and coworkers [2 (link)]. Ten control animals were fed with a regular rodent Chow diet (Purina #5001). After the two days under HC and Chow diets, five animals were sacrificed.
Hepatocytes were isolated from the rest of the HC and Chow mice by the two-step collagenase perfusion, as we previously described [12 (link)]. The viability was >90% as assessed by trypan blue exclusion. Hepatocytes were seeded at 2.13 × 105 cells per cm2 either in Lab-Tek chambered slides or in 10 cm dishes (Nalge, Nunc) in the Ham's F-12/Dulbecco's modified Eagle's basal hepatocyte growth medium supplemented with 10% fetal bovine serum. After 4 h of cells attachment, media were replaced by a serum-free basal hepatocyte growth medium. In the following day, cells were treated with 50 ng/mL HGF.
Twenty mice were randomly divided in two groups; in HC group 10 animals were fed with a high-cholesterol diet (HC, 2% cholesterol and 0.5% sodium cholate) for two days, as reported by Marí and coworkers [2 (link)]. Ten control animals were fed with a regular rodent Chow diet (Purina #5001). After the two days under HC and Chow diets, five animals were sacrificed.
Hepatocytes were isolated from the rest of the HC and Chow mice by the two-step collagenase perfusion, as we previously described [12 (link)]. The viability was >90% as assessed by trypan blue exclusion. Hepatocytes were seeded at 2.13 × 105 cells per cm2 either in Lab-Tek chambered slides or in 10 cm dishes (Nalge, Nunc) in the Ham's F-12/Dulbecco's modified Eagle's basal hepatocyte growth medium supplemented with 10% fetal bovine serum. After 4 h of cells attachment, media were replaced by a serum-free basal hepatocyte growth medium. In the following day, cells were treated with 50 ng/mL HGF.
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