Saos-2 cells were incubated with McCoy’s 5a culture medium contained in T75 culture flasks (Corning®, USA). McCoy’s 5a culture medium supplied with 2 mM L-glutamine, 100 units/mL penicillin G sodium, 250 ng/mL amphotericin B, 100 units/mL streptomycin sulphate, and 10% fetal bovine serum was used to grow human bone osteosarcoma cells (Saos-2). The culture medium was transferred to T75 culture flasks (Corning®, USA) and Saos-2 cells were seeded on it in a humidified air (95%) saturated with 5% CO2 at 37°C and kept for 72h in Corning® 96-well tissue culture plates till reaching a concentration of 5×104 cell/well.
T75 culture flask
Manufactured by Corning
Sourced in United States, Germany
The T75 culture flask is a cell culture labware product designed for the cultivation of adherent cells. It provides a surface area of 75 cm² for cell growth and proliferation. The flask is made of transparent, high-quality polystyrene material and features a vented cap to allow gas exchange.
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Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Thp 1, by American Type Culture Collection (2 mentions)
Rpmi 1640 medium, by Lonza (2 mentions)
Bovine serum, by Thermo Fisher Scientific (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
F0926, by BD (2 mentions)
B260300, by BD (2 mentions)
Glutamax 1, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Merck Group (1 mentions)
Dnase, by Merck Group (1 mentions)
Mccoy s 5a medium, by American Type Culture Collection (1 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (1 mentions)
Ht 29, by American Type Culture Collection (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Y 27632, by Merck Group (1 mentions)
Dimethyl sulphoxide dmso, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Rifampicin, by Merck Group (1 mentions)
41 protocols using t75 culture flask
1
Culturing Saos-2 Bone Osteosarcoma Cells
2
Isolation of Primary Murine Microglia
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Primary cultures of microglial cells were prepared from brains of newborn C57BL/6 mice (1–3 days) [19 (link)]. After removal of the meninges, cells were mechanically dissociated and suspended in Dulbecco’s modified Eagle’s medium (DMEM) with Glutamax I (Gibco, Karlsruhe, Germany) supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were plated at a density of two brains per T75 culture flask (Corning Costar GmbH, Wiesbaden, Germany) and incubated at 37 °C in a humid atmosphere with 5% CO2. Culture medium was changed twice a week. After 10–14 days, microglial cells were separated from the confluent astrocyte layer by shaking 200×/min for 30 min and plated in 96-well plates at a density of 50,000 cells/well for phagocytosis experiments. For staining, microglial cells were seeded on coverslips placed in a 24-well plate at a density of 50,000 cells/well.
3
Isolation of Primary Microglial Cells
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Primary cultures of microglial cells were prepared from brains of newborn C57/Bl6N mice (p0-p2). After removal of the meninges, cells were mechanically disrupted, treated with trypsin (Sigma-Aldrich, Taufkirchen, Germany) for 10 minutes to isolate the cells, afterwards treated with DNAse (Sigma-Aldrich, Taufkirchen, Germany), centrifuged for 10 minutes at 900 rpm at 4°C and suspended in DMEM supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were plated at a density of two brains per T75 culture flask (Corning Costar, Wiesbaden, Germany) and incubated at 37°C with 5% CO2. Microglial cells were isolated by shaking 200x/minute for 30 minutes and plated in 96-well plates at a density of 50,000 cells/well.
4
Culturing HT-29 Colorectal Adenocarcinoma Cells
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The female human colorectal adenocarcinoma intestinal epithelial cell (IEC) line HT-29 (ATCC, Manassas, VA, USA) was seed in T75 culture flask (Corning, Tewksbury, MA, USA) with McCoy's 5A medium (ATCC, Manassas, VA, USA) containing 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin (P/S; original solution at 10,000 U/ml) (Gibco, Carlsbad, CA, USA). Cells were split every 4-6 days when reaching 90-95% confluence using Trypsin-EDTA (Gibco, Carlsbad, CA, USA). No cell authentication was performed on the HT-29 cell line.
5
Intestinal Epithelial Sub-Culturing with ISEMF-Conditioned Media
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For intestinal epithelial sub-culturing, several experiments were initiated with crypt monocultures in the presence of ISEMF-conditioned media. ISEMF-conditioned Complete Crypt Medium (IC-CCM) was prepared in the following manner. ISEMFs were sub-cultured into a T-75 culture flask (Corning, Tewksbury, MA) and treated with ISEMF medium. The media was removed after one week of incubation and stored in a 4°C refrigerator for subsequent use. IC-CCM was constituted prior to each use by preparing a 1∶1 mixture of the spent ISEMF medium with doubly-concentrated CCM, such that the final concentration of the specific nutrients and growth factors above was unchanged. 2.5 µM CHIR99021 (Stemgent, Cambridge, MA) and 10 µM Y-27632 (Sigma) were also added.
Small intestinal crypts were isolated and suspended as above. They were then plated into 48-well Nunclon Delta-treated cell culture plates (Thermo Scientific, Waltham, MA) and treated with IC-CCM. The resultant epithelial units were sub-cultured every 4–5 days with a 1∶2 split ratio, using type XI-S collagenase (Sigma) and gentle mechanical disruption. They were then resuspended in collagen gel and re-plated followed by continued IC-CCM treatment.
Small intestinal crypts were isolated and suspended as above. They were then plated into 48-well Nunclon Delta-treated cell culture plates (Thermo Scientific, Waltham, MA) and treated with IC-CCM. The resultant epithelial units were sub-cultured every 4–5 days with a 1∶2 split ratio, using type XI-S collagenase (Sigma) and gentle mechanical disruption. They were then resuspended in collagen gel and re-plated followed by continued IC-CCM treatment.
6
HepG2 Cell Culture Protocol
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Human hepatoblastoma cell line, HepG2 (López-Terrada et al., 2009 ) was maintained in T75 culture flask (Corning, USA) in RPMI-1640 medium (Gibco, Thermo Fisher Scientific, Inc., MA, USA), supplemented with 10% heat-inactivated bovine serum (Gibco, MA, USA), 1× penicillin-streptomycin (Gibco, MA, USA) and 1× sodium pyruvate (GE Healthcare Life Sci., UT, USA) in an incubator at 37 °C with 5% CO2 supply. Dimethyl sulphoxide (DMSO; Sigma, Germany) was used as carrier to prepare stocks of plants extracts or compounds as well as negative control. Rifampicin (Sigma, Germany) was used as standard PXR-mediated CYP3A4 inducer or positive control.
7
Isolation and Culture of Umbilical Cord MSCs
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The umbilical cord samples from cesarean section were collected under sterile conditions, cleaned with phosphate buffered saline (PBS), and cut into 1 mm3 pieces. Then, the tissue blocks were placed into culture bottles at a distance of 0.5 cm. After slowly adding MSCs’ medium (7501, Sciencell, USA), a T75 culture flask (430641, Corning, USA) was placed in an incubator at 37°C. The cells gradually emerged after 5-7 days. The medium was changed every 3-5 days. When the fusion degree was 70-80%, the cells were digested and passaged with 0.5% trypsin. After passaging to the fifth generation, the cells were identified and subsequently tested. Sample collection was approved by the ethics committee of Peking University First Hospital (No. 2020 [279]), and each patient signed an informed consent form.
8
Cytotoxicity Evaluation of Fdb in Cell Lines
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LLC-MK2 and K562 cells were grown to 95% confluence in a T75 culture flask (Corning®, Corning, NY, USA). The cells were plated onto a 96-well plate (Corning®, New York, NY, USA) (100 µL per well for 10,000 cells) and cultured for 48 h. The cells were treated with a 10-fold diluted concentration series (100.0, 10.0, 1.0, 0.1, and 0.01 µM) of Fdb in culture media plus 0.0001% DMSO. The non-treatment control group was treated with culture media plus 0.0001% DMSO. The treated cells were incubated at 3 °C in a 5% CO2 incubator for 24 and 72 h (for LLC-MK2 cells) or 96 h (for K562 cells). MTT solution was added (1:10 by volume) and incubated until the purple formazan crystal formed. The crystal product was solubilized by MTT solubilizing agent (80% isopropanol and 10% Triton X-100 in 1.0 N HCl). The product was measured in terms of optical absorbance at 562 nm by a microplate spectrometer. The data were quantified and a inhibition concentration index of 50% cell viability (IC50) was determined by non-linear regression analysis using GraphPad® Prism 5 software.
9
Differentiation of THP-1 Cells into Macrophages
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The human monocytic leukemia cell line (THP‐1; American Type Culture Collection, Rockville, MD, USA) was cultured in RPMI 1640 medium (Lonza, Basel, Switzerland) containing 10% of fetal bovine serum (FBS; Hyclone PerBio, Etten‐Leur, The Netherlands) and 1% of penicillin/streptomycin (Invitrogen) at 37 ˚C under 5% of CO2. Cells were sub‐cultured twice per week and set at 0.25 × 106 mL−1 in 20 mL medium in a T75 culture flask (Corning, Amsterdam, The Netherlands). To differentiate the cells into macrophages, 0.5 × 106 cells were exposed to 100 ng mL−1 phorbol 12‐myristate 13‐acetate (PMA; Sigma‐Aldrich) in 0.5 mL RPMI medium for 72 h in 24‐well cell culture plate (Greiner Bio‐one). Following extensive washing to remove residual PMA, the cells were rested for 72 h. Rested THP‐1 macrophages were exposed to medium (supplemented RPMI) as control or a homogeneous chitin suspension in medium, achieved by thorough mixing through vortexing and resuspending, at a concentration of 0.1 mg mL−1 and incubated at 37 ˚C for 24 h.
10
Isolation of NSC-Derived Extracellular Vesicles
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For the isolation of NSC-derived EVs, frozen vials containing passage 11 NSCs were thawed at 37°C and plated on to a T-75 culture flask (Corning) and grown at 37°C in a CO2 incubator. Following 70% confluency, the cells were dislodged using 1 U/ml of dispase (Gibco), washed with NSC media (Gibco), and seeded at ~500 cells per cm2 into 150 × 20 mm diameter tissue culture plates (Corning) in NSC expansion medium. Once NSCs reached 90% confluency, the media was harvested and used for isolating EVs or stored at −80°C for further use.
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