Adipose tissues were obtained from the subcutaneous abdominal fat and digested with collagenase I at 37°C for 45 min with continuous shaking. After neutralizing enzyme activity with L-DMEM (low glucose-Dulbecco's modified Eagle medium) supplemented with 10% FBS (Clark, USA), the homogenate was filtered and centrifuged, and the ADSCs were suspended in L-DMEM supplemented with 10% FBS, 2 mM L-glutamine, and 100 μg/ml penicillin and streptomycin (Solarbio, China). The cells were cultured at 37°C under 5% CO2 in a humidified incubator (Galaxy 170 S, Eppendorf, Germany). The ADSCs were characterized as previously described [31 (link)], cultured in serum-free L-DMEM for 48 h. Then, the medium was aspirated and centrifuged at 1000 g for 15 min at 4°C to remove the cell debris. The supernatant was then centrifuged at 5000 g for 50 min at 4°C using 3 kDa MWCO (Millipore, Billerica, USA) to concentrate it by 25-fold. The CM aliquots were transferred to sterile 1.5 ml EP tubes and stored at -80°C.
L dmem
Manufactured by Solarbio
Sourced in United States, China
L-DMEM is a type of cell culture medium that is commonly used for the in vitro cultivation of cells. It provides a balanced salt solution, amino acids, vitamins, and other essential nutrients required for cell growth and maintenance.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Solarbio (2 mentions)
Penicillin and streptomycin, by Solarbio (1 mentions)
Galaxy 170, by Eppendorf (1 mentions)
Fetal bovine serum (fbs), by Clark Bioscience (1 mentions)
3 kda mwco, by Merck Group (1 mentions)
L dmem, by Clark Bioscience (1 mentions)
L glutamine, by Solarbio (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Heracell 150i, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin mixture, by Solarbio (1 mentions)
3 protocols using l dmem
1
Isolation and Characterization of ADSCs
2
Isolation and Culture of Rabbit BMSCs
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Following a previously published protocol (23 ), a 2-week-old New Zealand rabbit was used for the collection of BMSCs. Rabbits were anesthetized by intravenous injection of 30 mg/kg pentobarbital sodium into the ear margin. A bone marrow puncture needle was then inserted into the posterior superior iliac spine of the rabbit. A total of 20 ml of low molecular weight heparin calcium (Tianjin Chase Sun Pharmaceutical Co., Ltd.) was injected to extract the bone marrow. The inner wall of the syringe was moistened with low molecular weight heparin 5 min before extraction. The extracted bone marrow was then injected into a sterile centrifuge tube and was diluted 1:5 with L-DMEM (Beijing Solarbio Science & Technology Co., Ltd.). The bone marrow-DMEM mixture was centrifuged at 1,200 x g for 5 min at 25˚C. The supernatant was discarded and subsequently cells were resuspended in 20 ml DMEM containing 10% fetal calf serum (Gibco; Thermo Fisher Scientific, Inc.). The extracted cells were added to a culture flask and placed in an incubator at 37˚C and 5% CO2. The medium was replaced with fresh medium 48 h later. The full-marrow adherent method (24 (link)) was employed for separation and purification of rabbit BMSCs. The culture medium was replaced every 3 days until the cells reached 80% confluency, when they were passaged for further experiments.
3
Evaluating Collagen Hydrogel Cytotoxicity
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The cytotoxicity of collagen hydrogels was carried out as reported by Fiocco et al. [70 (link)] with slight modification. Aseptic collagen hydrogels were prepared and lixiviated with L-DMEM (HyClone, Logan, UT, USA) for 72 h at 37 °C with lixiviation ratio as 0.1 % (w/v). The Cell density of NIH-3T3 fibroblasts was adjusted to 1 × 104 /well, and cultured in a 96-well plate at 37 °C, 5% CO2 (HERAcell 150i, Thermo Scientific, USA). After 24 hours of culture, the culture medium of experimental groups was replaced with the lixiviation solution, containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin mixture (100 unit/mL penicillin, 100 μg/mL streptomycin) (Solarbio, Beijing, China), and the control group was replaced with L-DMEM containing 10% FBS and 1% penicillin-streptomycin mixture. The percentage of viable cells was detected using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT method after 24 h, 48 h, and 72 h of continuous culture. The cell viability was calculated as follows:
where A0, At and A were the absorbance at 490 nm of control group, experimental group and the group without cells, respectively.
where A0, At and A were the absorbance at 490 nm of control group, experimental group and the group without cells, respectively.
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