Fresh animal skin specimens were obtained from C57BL6 mouse aged from 6 to 8 weeks. Mouse were killed and placed on 70% alcohol for 15 min. The specimens were washed with sterile phosphate-buffered saline (PBS), and the subcutaneous tissues were carefully removed. Then the skins were cut into small pieces (1–2 mm3). The skin specimens were digested with 0.1% Dispase (Sigma, United States) at 4°C overnight, the epidermal layers were removed and the remaining dermal parts were further digested with 0.1% collagenase I (Sigma, United States) at 37°C for another 4 h. The digested tissues were centrifuged and suspended in high-glucose DMEM (Bio Idea, Tehran, Iran). Tissue fragments were cultured in DMEM supplemented with 10% FBS (Bio Idea, Tehran, Iran), 300 μg/m1 L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Bio Idea, Tehran, Iran) for about 7 days until Fibroblasts grew out of the tissue. Then, cells were removed and cultured in T25 flask at 37°C in a humidified atmosphere with5% CO2. When cultured cells reached more than 90% of confluence, the culture medium were removed and replaced with FBS free medium. These cells were divided into three groups: group 1 (control, no treatment), group 2 (cells treated with 10 mM ammonium lactate for 48 h) and group 3 (irradiated cells). The cell culture supernatant was collected from each group after 48 h.
L glutamine
Manufactured by Bioidea
L-glutamine is a common amino acid found in the human body. It is a key component in the synthesis of proteins and plays a crucial role in various metabolic processes.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Bioidea (2 mentions)
Penicillin, by Bioidea (2 mentions)
Fetal bovine serum (fbs), by Bioidea (2 mentions)
Pkh26 red uorescent cell linker kit, by Merck Group (2 mentions)
Collagenase 1, by Merck Group (1 mentions)
Dispase, by Merck Group (1 mentions)
Sz 100, by Horiba (1 mentions)
Pkh26 red fluorescent cell linker kit, by Merck Group (1 mentions)
Zen0040, by Malvern Panalytical (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
5 protocols using l glutamine
1
Isolation and Culture of Mouse Dermal Fibroblasts
2
Extracellular Vesicle Internalization in MG63 Cells
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MG63 cell line was cultured in the presence of RPMI containing 10% FBS (Gibco), 1% L-glutamine (Bioidea), and 1% penicillin/streptomycin (Gibco) at 37 °C and 5% CO 2 . ECVs were labeled with PKH26, using PKH26 red fluorescent cell linker kit (Sigma) according to the manufacturer's protocol. Briefly, 0.5 mL of ECVs mixed with 498 µL of dilution buffer and 2 µL PKH26 and incubated for 5 min and diluted with FBS at a ratio of 50/50. Then, the mixture was centrifuged at 100 000 g for 90 minutes at 4 °C. The pellets were mixed with 1 mL of the culture medium. Labeled ECVs were added to MG63 cell line cultures and incubated for 22 hours. Finally, the internalization of the labeled ECVs was evaluated by fluorescence microscopy. For negative control, the same amount of dilution solution without ECVs was added to the cells.
Dynamic Light Scattering (DLS)
ECV size was evaluated by nanoparticle analyzer (SZ-100 Horiba, Japon) equipped with a 532-nm wavelength, 10 mW power, and operating at an angle of 173. ECVs were transferred to cuvettes (ZEN0040, Malvern, Herrenberg, Germany). The measurements were made at a fixed position and at 25 °C. Three independent measurements were performed for each sample, and three samples were analyzed, and the mean value calculated.
Dynamic Light Scattering (DLS)
ECV size was evaluated by nanoparticle analyzer (SZ-100 Horiba, Japon) equipped with a 532-nm wavelength, 10 mW power, and operating at an angle of 173. ECVs were transferred to cuvettes (ZEN0040, Malvern, Herrenberg, Germany). The measurements were made at a fixed position and at 25 °C. Three independent measurements were performed for each sample, and three samples were analyzed, and the mean value calculated.
3
ECV Internalization in MG63 Cells
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MG63 cell line was cultured in the presence of RPMI containing 10% FBS (Gibco), 1% L-glutamine (Bioidea), and 1% penicillin/streptomycin (Gibco) at 37°C and 5% CO 2 . ECVs were labeled with PKH26, using PKH26 red uorescent cell linker kit (Sigma) according to the manufacturer's protocol. Brie y, 0.5 mL of ECVs mixed with 498 µL of dilution buffer and 2µL PKH26 and incubated for 5 min and diluted with FBS at a ratio of 50/50. Then, the mixture was centrifuged at 100 000g for 90 minutes at 4°C. The pellets were mixed with 1mL of the culture medium. Labeled ECVs were added to MG63 cell line cultures and incubated for 22 hours. Finally, the internalization of the labeled ECVs was evaluated by uorescence microscopy. For negative control, the same amount of dilution solution without ECVs was added to the cells.
4
Extracellular Vesicle Uptake in MG63 Cells
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MG63 cell line was cultured in the presence of RPMI containing 10% FBS (Gibco), 1% L-glutamine (Bioidea), and 1% penicillin/streptomycin (Gibco) at 37 °C and 5% CO 2 . ECVs were labeled with PKH26, using PKH26 red uorescent cell linker kit (Sigma) according to the manufacturer's protocol. Brie y, 0.5 mL of ECVs mixed with 498 µL of dilution buffer and 2 µL PKH26 and incubated for 5 min and diluted with FBS at a ratio of 50/50. Then, the mixture was centrifuged at 100 000 g for 90 minutes at 4 °C. The pellets were mixed with 1 mL of the culture medium. Labeled ECVs were added to MG63 cell line cultures and incubated for 22 hours. Finally, the internalization of the labeled ECVs was evaluated by uorescence microscopy. For negative control, the same amount of dilution solution without ECVs was added to the cells.
5
Culturing Human Colorectal Cancer Cell Line
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The human colorectal adenocarcinoma cell line LS174T was obtained from the National Cell Bank of Iran (Pasteur Institute, Tehran, Iran) and was maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY, USA) with 4.5 g/L glucose, supplemented with 10% fetal bovine serum (FBS; Bio-IDEA, Tehran, Iran), 2-mM l-glutamine, 100 IU/mL penicillin, and 100 µg/mL streptomycin (Bio-IDEA). The cells were cultured to their exponential growth phase. LS174T cells were harvested using trypsin-EDTA and incubated at 37°C with 5% CO 2 .
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