Human bone marrow-mesenchymal stem cells (hBM-MSCs) were purchased from Cell Engineering For Origin (CEFO) (Korea) and cultured in T75 flasks (Falcon; Corning, USA) according to the supplier’s recommendations. hBM-MSCs were cultivated in mesenchymal stem cell growth medium (Gibco, Grand Island, NY, USA) as previously described (Jeong and Cho, 2015 (link)). Passage seven (P-7) hBM-MSCs were used. Rat fetal neural stem cells (NSCs) were purchased from Gibco (Grand Island, NY, USA) and cultured in T75 flasks (Falcon) according to the supplier's recommendations. NSCs were cultivated in growth medium containing 2% StemPro® NSC supplement (Gibco, Grand Island, NY, USA), 2 mM GlutaMaxTM I supplement (Gibco, Grand Island, NY, USA), 20 ng/ml basic fibroblast growth factor (bFGF) (Gibco, Carlsbad, CA, USA), and 20 ng/ml epidermal growth factor (Gibco, Carlsbad, CA, USA) in KnockOutTM Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY, USA). P-3 NSCs were used for the experiments. Human osteosarcoma cells (U2OS) were cultured in DMEM/F12 (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (FBS) (Origin Canada; Gibco, Grand Island, NY, USA), 2 mM L-glutamine, and 1% penicillin/streptomycin antibiotics at 37°C in a 5% CO2 incubator.
T75 flask
Manufactured by BD
Sourced in United States, United Kingdom, Switzerland
The T75 flasks are standard cell culture flasks with a growth surface area of 75 cm2. They are designed for the cultivation and expansion of adherent cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (16 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (9 mentions)
T25 flask, by BD (6 mentions)
Dnase 1, by Thermo Fisher Scientific (6 mentions)
Trypsin edta, by Thermo Fisher Scientific (6 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
Trypsin, by Thermo Fisher Scientific (5 mentions)
Dmem f12, by Thermo Fisher Scientific (4 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (4 mentions)
Caco 2, by Merck Group (3 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (2 mentions)
Mimic cytokine mix, by Thermo Fisher Scientific (2 mentions)
Tnf α, by Thermo Fisher Scientific (2 mentions)
Il 1β, by Thermo Fisher Scientific (2 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (2 mentions)
Z2 cell counter, by Beckman Coulter (2 mentions)
57 protocols using t75 flask
1
Culturing Human and Rat Stem Cells
2
NCCIT Cell Culture and RNA Extraction
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The NCCIT cells are cultured in RPMI growth medium supplemented with 10% v/v foetal bovine serum, 1% v/v penicillin-streptomycin and 2 mM L-glutamine. They are allowed to grow to 100% confluence in a T75 flask (Falcon, VWR UK). The cells were typically sub-cultured twice a week using trypsin-EDTA to dislodge them from the substratum. The cells were used to seed a 12 well cluster plate at 5 × 105 cells/well and the next day they were treated with IL-1β (100 pg/mL). The cells were harvested 24 h post-treatment by centrifugation and the cell pellets were stored at −80 °C prior to RNA extraction. Total RNA was extracted using the Qiagen QIAshredder (79656) and the Qiagen RNeasy Mini kit (74106) according to the manufacturer’s protocol.
3
Cell Culture Protocols for C3H10T1/2 and RAW 264.7
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C3H10T1/2 and RAW 264.7 cells were grown in a T75-flask (BD Falcon, USA) in a 5% CO2 humidified atmosphere at 37 °C. The C3H10T1/2 cells (clone 8 American Type Culture Collection, USA) and RAW 264.7 were cultured in α-MEM, supplemented with 10% Fetal Bovine Serum (FBS) (HyClone Laboratories Inc. USA), l -glutamine, and 1% antibiotics (50 U/mL penicillin and 50 μg/mL streptomycin). After reaching a subconfluent state, C3H10T1/2 cells were trypsinized with 1 × trypsin–EDTA and plated into 48 or 96-well culture plates based on experimental need; the medium was then changed every other day.
4
Vesicular Stomatitis Virus Propagation in BHK-21 Cells
5
In Vitro HUVEC Culture on Collagen Films
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All experimental procedures complied with the act on life ethics and safety of
the Ministry of Health and Welfare of South Korea. HUVECs were obtained from
Invitrogen (Carlsbad, CA, USA) and used in passages 5 or 7. The cells were
cultured in Medium 200 (Gibco) with low-serum growth supplement (Gibco)
containing 20% fetal bovine serum (Gibco) and 1% penicillin-streptomycin
(HyClone). The cells were cultured in a T75 flask (Falcon) under a humidified
atmosphere of 5% CO2 at 37°C. Trypsin/ethylenediaminetetraacetic
acid solution was used to detach cells from the culture flask. The
surface-treated cover glass (collagen film or denatured collagen film) was
placed in a cell culture dish (diameter = 35 mm), loaded with
106 cells/mL cell suspension, and incubated under a humidified 5%
CO2 atmosphere at 37°C. Figures1 A and 1 B illustrate the ECs cultured on the
collagen and denatured collagen films, respectively. HUVECs were cultured on the
collagen film or denatured collagenase-treated collagen film for 1, 3, 6, 12,
and 24 h. For DHM imaging, the test samples were fixed with 4%
paraformaldehyde solution for 20 min at room temperature, and washed with
PBS solution. Each sample was placed on a drop of PBS solution at the center of
a polydimethylsiloxane chamber. Sample images were photographed at room
temperature.
the Ministry of Health and Welfare of South Korea. HUVECs were obtained from
Invitrogen (Carlsbad, CA, USA) and used in passages 5 or 7. The cells were
cultured in Medium 200 (Gibco) with low-serum growth supplement (Gibco)
containing 20% fetal bovine serum (Gibco) and 1% penicillin-streptomycin
(HyClone). The cells were cultured in a T75 flask (Falcon) under a humidified
atmosphere of 5% CO2 at 37°C. Trypsin/ethylenediaminetetraacetic
acid solution was used to detach cells from the culture flask. The
surface-treated cover glass (collagen film or denatured collagen film) was
placed in a cell culture dish (diameter = 35 mm), loaded with
106 cells/mL cell suspension, and incubated under a humidified 5%
CO2 atmosphere at 37°C. Figures
collagen and denatured collagen films, respectively. HUVECs were cultured on the
collagen film or denatured collagenase-treated collagen film for 1, 3, 6, 12,
and 24 h. For DHM imaging, the test samples were fixed with 4%
paraformaldehyde solution for 20 min at room temperature, and washed with
PBS solution. Each sample was placed on a drop of PBS solution at the center of
a polydimethylsiloxane chamber. Sample images were photographed at room
temperature.
6
Adipose-Derived Stem Cell Isolation
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For stem cell isolation, adipose tissue was collected from bilateral inguinal fat pads and epididymes of BN rats. On average, 45 g of fat tissue was harvested from three rats. Enzymatic fat digestion was performed by collagenase type II (Worthington Biochemical Corp, Lakewood, NJ, USA) and bovine serum albumin (Millipore, Billerica, MA, USA) in Hanks’ balanced saline solution (Cellgro Mediatech Inc., Manassas, VA, USA) for 60 min at 37°C. The digested tissue was centrifuged at 1,000 rpm for 10 min. The cellular pellet [stromal vascular fraction (SVF)] was resuspended in erythrocyte lysis buffer and filtered with sterile gauze. SVF was transferred to sterile culture flasks with Dulbecco’s modified Eagle’s medium (DMEM; Cellgro Mediatech, Inc.) plus supplemental Ham’s F-12 medium (Gibco, Grand Island, NY, USA). After overnight incubation for cell attachment, non-adherent cells were removed using a phosphate buffered saline wash. The attached ASCs were cultured in DMEM/F-12 supplemented with 10% fetal bovine serum (ATLAS Biologicals, Fort Collins, CO, USA), 0.1 μM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), 1% penicillin–streptomycin (Gibco), and 1.25 mg/L amphotericin B (Gibco). SVF from 45 g of fat tissue was plated in a T75 flask (BD Falcon). Approximately, 5 × 105 confluent ASCs were obtained (passage 0). ASCs were expanded in vitro until passage 3.
7
Culturing HUVECs and Transfecting HEK293 Cells
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HUVECs obtained from Lonza (cat. #C2519) were cultured by the same procedure as previously described (Yazbeck et al., 2017 (link)). Briefly, cells were plated in a T-75 flask (BD Falcon) coated with 0.1% gelatin and cultured in EBM-2 media (Lonza) supplemented with growth factors and 10% FBS (Thermo Fisher Scientific). Cells were cultured in a 37°C humidified incubator in the presence of 5% CO2 and 95% O2 until they formed a monolayer and achieved the desired confluence. HUVECs between passages 5 and 6 were used for these studies. The HEK293 cell line (American Type Culture Collection) was cultured in DMEM (Gibco) supplemented with 10% FBS (Thermo Fisher Scientific) and 5% penicillin/streptomycin (Thermo Fisher Scientific). HEK293 cells were transfected with indicated cDNAs using FuGENE HD (Roche).
8
Culturing Human Dermal Fibroblasts
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Primary cultured human dermal fibroblasts were obtained from consenting non-diabetic patients undergoing major lower limb amputations from intact skin at the proximal margin.
Skin samples were immediately placed in a culture medium of Dulbecco's modified Eagle's medium 4.5g/dL glucose (25mM), (DMEM, catalog number (cat. no.) 31966-021; Invitrogen, Paisley, UK), 10% fetal calf serum (GIBCOs, Paisley, UK), 100U/ml penicillin and 100g/ml streptomycin. The dermis and epidermis were separated using sharp dissection, and dermal cells seeded in a T75 flask (Falcon, Franklin Lakes, NJ, USA) with 25ml of culture medium and incubated in a humidified atmosphere of 21% O2 and 5% CO at 37°C. At passages 2 to 3, all cells were transferred to a low glucose media (5.5mM), and further cultured for at least two weeks.
Skin samples were immediately placed in a culture medium of Dulbecco's modified Eagle's medium 4.5g/dL glucose (25mM), (DMEM, catalog number (cat. no.) 31966-021; Invitrogen, Paisley, UK), 10% fetal calf serum (GIBCOs, Paisley, UK), 100U/ml penicillin and 100g/ml streptomycin. The dermis and epidermis were separated using sharp dissection, and dermal cells seeded in a T75 flask (Falcon, Franklin Lakes, NJ, USA) with 25ml of culture medium and incubated in a humidified atmosphere of 21% O2 and 5% CO at 37°C. At passages 2 to 3, all cells were transferred to a low glucose media (5.5mM), and further cultured for at least two weeks.
9
C3H10T1/2 and RAW 264.7 Cell Culture
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C3H10T1/2 and RAW 264.7 cells were grown in a T75-flask (BD Falcon, USA) in a 5% CO 2 humidified atmosphere at 37°C. The C3H10T1/2 cells (clone 8 American Type Culture Collection, USA) and RAW 264.7 were cultured in α-MEM, supplemented with 10% Fetal Bovine serum (FBS) (HyClone Laboratories Inc. USA), L-glutamine and 1% antibiotics (50 U/mL penicillin and 50mg/mL streptomycin). After reaching a subconfluent state, C3H10T1/2 cells were trypsinized with 1x trypsin-EDTA and plated into 48 or 96-well culture plates based on experimental need; the medium was then changed every other day.
10
Murine 4T1 Breast Cancer Model
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