Human skin fibroblasts were isolated from 3-mm skin punch biopsy (n = 15 for RTT and n = 15 for controls). Cells were cultured in DMEM (Sigma-Aldrich, Milan, Italy), containing 20% fetal calf serum [9] (link) (FCS, Sigma-Aldrich) and antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin) (Lonza, Milan, Italy). Cells were incubated at 37 C° in a humidified atmosphere at 5% CO2 for 3 days. When fibroblasts growing from the dermal pieces formed a confluent layer, the dermal pieces were removed and trypsin/EDTA mixture (Sigma-Aldrich) was added to separate fibroblasts. Cells were transferred to 25-cm2 culture flasks (Falcon, Perugia, Italy) and subcultured in 10% FCS/DMEM. Fibroblasts from passage 3 to 5 were used for the experiments. 1×106 cells were seeded in each flask (25 cm2), whereas the experiments were performed when cells reached 70/80% confluence.
Trypsin edta mixture
Manufactured by Merck Group
Trypsin/EDTA mixture is a laboratory reagent used for cell dissociation and harvesting. Trypsin is a proteolytic enzyme that cleaves peptide bonds, enabling the detachment of adherent cells from culture surfaces. EDTA is a chelating agent that aids in the disruption of cell-cell and cell-matrix interactions. This combination of trypsin and EDTA is commonly used to facilitate the dissociation and harvesting of a variety of cell types for further experimentation or subculturing.
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Lab products found in correlation
Dulbecco modified eagle medium (dmem), by BD (1 mentions)
25 cm2 culture flasks, by BD (1 mentions)
Fetal calf serum (fcs), by BD (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Penicillin, by Lonza (1 mentions)
Streptomycin, by Lonza (1 mentions)
2 protocols using trypsin edta mixture
1
Isolation and Culture of Human Skin Fibroblasts
2
Establishing a Rat BMEC-based BBB Model
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Brain microvascular endothelial cells (BMECs) were extracted from 3-week-old rats as a described in a previous study. [24] Brie y, BMECs were cultured in DMEM/F12 with puromycin at 37 °C with a humidi ed atmosphere of 5% CO 2 /95% air for 2 d. On the 3rd day, the puromycin was removed from the medium.
When the cells reached 80% con uence (5th - 6th day), the endothelial cells were passed using a trypsin/EDTA mixture (Sigma). The cells were transferred to 12-well Transwell inserts coated with collagen type IV and bronectin to form the BBB model in vitro. The endothelial monolayer showed positive expression of the endothelial marker von Willebrand factor (supplementary Fig. 1) after immunostaining. BMECs were incubated at 37 °C for 6 h. The cells were divided into three groups (each group, n = 3): the normoxia (21% O 2 ), hypoxia (1% O 2 + 5% CO 2 + 94% N 2 ) and hypercapnia groups (1% O 2 + 15% CO 2 + 84% N 2 ).
When the cells reached 80% con uence (5th - 6th day), the endothelial cells were passed using a trypsin/EDTA mixture (Sigma). The cells were transferred to 12-well Transwell inserts coated with collagen type IV and bronectin to form the BBB model in vitro. The endothelial monolayer showed positive expression of the endothelial marker von Willebrand factor (supplementary Fig. 1) after immunostaining. BMECs were incubated at 37 °C for 6 h. The cells were divided into three groups (each group, n = 3): the normoxia (21% O 2 ), hypoxia (1% O 2 + 5% CO 2 + 94% N 2 ) and hypercapnia groups (1% O 2 + 15% CO 2 + 84% N 2 ).
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