All cell lines were cultured at 37°C in a 5% CO2 incubator. Cell lines GES-1, BGC-823, AGS, and SGC-7901 were purchased from CCTCC (China Center for Type Culture Collection). GES-1 and SGC-7901 were maintained in DMEM (Gibco, Invitrogen, USA) supplemented with 10% fetal bovine (Gibco, Invitrogen, USA). AGS and BGC-823 were maintained in RPMI medium 1640 (Gibco, Invitrogen, USA) supplemented with 10% fetal bovine (Gibco, Invitrogen, USA).
Fetal bovine
Manufactured by Thermo Fisher Scientific
Sourced in United States
Fetal bovine serum (FBS) is a biological material derived from the blood of bovine fetuses. It is a commonly used supplement in cell culture media, providing a source of growth factors, nutrients, and other components to support the growth and maintenance of cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (7 mentions)
Penicillin, by Thermo Fisher Scientific (6 mentions)
Streptomycin, by Thermo Fisher Scientific (6 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (6 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (4 mentions)
Trypsin, by Thermo Fisher Scientific (3 mentions)
Agarose, by Thermo Fisher Scientific (2 mentions)
Catheter based pressure transducer, by Millar (2 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (2 mentions)
Ultrasonic flow meter, by Transonic Systems (2 mentions)
Hek293t, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Psicheck 2 vector, by Promega (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Hct116, by American Type Culture Collection (1 mentions)
Sw480, by American Type Culture Collection (1 mentions)
Dld 1, by American Type Culture Collection (1 mentions)
Caco 2, by American Type Culture Collection (1 mentions)
Hcoepic, by American Type Culture Collection (1 mentions)
26 protocols using fetal bovine
1
Cell Culture Conditions for Gastric Cancer Lines
2
Luciferase Assay for miR-19a-3p Targeting
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The Human embryonic kidney 293 T (HEK-293 T) cells were obtained from Thermo Fisher Scientific and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Thermo Scientific) containing 10% fetal bovine (Gibco) and grown in 5% CO2 incubator at 37 °C. The reporter plasmids containing the sequence of the TGFBR3 3′-UTR with rs1804506 C allele or T allele were constructed and cloned into the NotI/XhoI restriction enzyme sites of the psiCHECK-2 vector (Promega). DNA sequencing was used to verify all the cloned sequences. For luciferase assays, 2.0 × 104 HEK-293 T cells in 100 μl growth medium were seeded in 96-well plates. Next, the has-miR-19a-3p mimics or its negative control (Oligobio) was co-transfected with constructed plasmids using Lipofectamine 2000 (Invitrogen). Forty-eight hours after transient transfection, the Dual Luciferase Reporter Assay System (Promega) was used to measure the luciferase activity according to the manufacturer’s instructions, and the Renilla luciferase activity was normalized to firefly luciferase activity. All assays were conducted in triplicate.
3
Colorectal Cancer Tissue RNA Analysis
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From September 2019 to December 2020, colorectal cancer tissues and paired adjacent noncancerous tissues of 62 patients were collected in the First Affiliated Hospital of Sun Yat-sen University. Our study was approved by the Ethics Committee of the First Affiliated Hospital of Sun Yat-Sen University. Before RNA extraction, the samples were stored in liquid nitrogen. Human normal colonic epithelial cells (HCoEpiC) and CRC cell lines (HCT116, DLD-1, SW480, LOVO, CaCo2, HT29) were obtained from American Type Culture Collection (ATCC, Manassas, VA, United States). Cells were cultured using DMEM (Gibco, CA, United States) containing 10% fetal bovine (Gibco, CA, United States), 100 U/ml penicillin, and 100 mg/ml streptomycin (Invitrogen, Carlsbad, CA, United States). All cell lines were cultured at 37°C with 5% CO2. Trizol reagent was used to extract total RNA from tissues and cell lines (Invitrogen, Carlsbad, CA). Quantitative real-time PCR (qPCR) was performed on the light cycle 480 real-time PCR platform (Merlan Roche, France) to measure gene expression. The qRT-PCR primer sequences are provided in Table 1 . The relative quantification values for RNA were calculated by the 2−ΔΔCt method.
4
Cell Culture of RCC Cell Lines
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The RCC cell lines (786-O and ACHN) were purchased from Shanghai Cell Bank, Chinese Academy of Sciences. Cells were maintained in RPMI 1640 Medium (Gibco, USA) supplemented with 10% fetal bovine (BI, Israel), 100 U/ml penicillin and 100μg/ml streptomycin (Gibco, USA). Cells were incubated in a humidified atmosphere of 95% air and 5% CO2 at 37℃.
5
Co-transfection of Sheep Fetal Fibroblasts
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A sheep fetal fibroblast (SFB) cell line (China Agricultural University) was cultured in the medium containing 10% fetal bovine (Gibco, USA) serum in 5% CO2 at 37 °C. The cells were seeded in 6-well plates (Thermo Scientific, USA). After 24 h, cells were co-transfected with a mixture of plasmid, pcDNA3.1(+)-Cas9, pMD-19T-U6-sgRNA, and tGFP expression vector with the mass ratio of 2:1:1 (2500 ng in total per well), following the instruction of Lipofectamine3000 (Life Technologies, USA). Cells were harvested 48 h after transfection.
6
Adaptive Phage Library for Antibody Screening
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U937 (human lymphoma monocytic cell line), A549 (human non-small cell lung cancer cell line) and anti-TNF-α ScFv were stored in the hematology laboratory of the 2nd Affiliated Hospital. Healthy human leukocytes were purchased from a blood bank in Xi'an. Cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine (Gibco) and cultured at 37°C in a 5% CO2 humidified incubator. ALPHA™ (Adaptive Library Panning for Human Antibody) is a 8.86*1010 human synthetic phage library (EUREKA, China).
7
Skeletal Stem Cell Culture Optimization
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Skeletal Stem Cells were cultured in alpha-MEM GlutaMax supplemented with 10% Fetal Bovine and 1% Penicillin–Streptomycin (Gibco-Life Technologies, Grand Island, NY, USA) after seeding on gelatin coated wells. Cells were incubated under low O2 conditions (2% atmospheric oxygen, 7.5% CO2) for 48 h and then moved to standard conditions (5% CO2).
8
Ex Vivo Arterial Perfusion Bioreactor
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Porcine common carotid arteries from large Yorkshire pigs (250-350 lbs.) were obtained from local and regional abattoirs. Vessels were then rinsed in saline and excess fat, connective tissue, and fascia removed from each artery and cut into approximately 8-cm segments. Arteries were stored in 15 mL falcon tubes at −20°C until needed.
To mimic cardiovascular flow conditions, an ex vivo bioreactor system was utilized that incorporated a computer-controlled gear pump and closed circuit hosted within a temperature-controlled incubator (Figure 1A ). The end of each artey was connected to a luer fitting and assembled into our bioreactor housing. The space between the artery and the protective outer sleeve was then filled with tissue-simulating agent (Agarose, Thermo Fisher Scientific). To measure pressure within the flow circuit, a catheter-based pressure transducer (Millar Instruments, Houston, TX, USA) is positioned within the flow circuit. The volumetric flow rate was quantified using an ultrasonic flow meter (Transonic Systems Inc., Ithaca, NY, USA). The circulating medium consisted of Dulbecco’s Modified Eagle’s Medium containing low glucose (1000 mg/L), 4.0 mmol/L L-glutamine, 110 mg/L sodium pyruvate, pyridoxine hydrochloride, 10% fetal bovine (Gibco), and 1% antibiotic-antimycotic (Gibco).
To mimic cardiovascular flow conditions, an ex vivo bioreactor system was utilized that incorporated a computer-controlled gear pump and closed circuit hosted within a temperature-controlled incubator (
9
RPE Explant Culture and Treatments
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RPE explant culture was performed as previously described [47 ]. Eyes were enucleated from euthanized 6-8 weeks old C57BL/6J, Ripk3−/−, Mlkl−/−, or Ripk3−/−/Mlkl−/− mice and washed with 1x PBS twice. After removing the connective tissue and anterior parts (cornea and lens), the eyecups were cut into four petals. Neural retinae were then peeled off and the RPE/Choroid/Sclera complexes were mounted on a PVDF membrane with the sclera attached to the membrane. RPE flat mounts were then transferred to a 24 well plate with 1 mL high glucose culture medium in each well, medium was supplemented with 10% fetal bovine (HyClone), 1% penicillin-streptomycin solution (HyClone), 1% MEM Non-Essential Amino Acids (Gibco, 11140-50), and 1% GlutaMAX (Gibco, 35050-061), with the RPE facing down and cultured in an incubator at 37 °C with 5% CO2. Tissues were primed with inhibitors (Lip-1 at 10 μM, Nec-1 at 300 μM, NSA at 5 μM) for 12 h followed by treatment with 4-HNE at 100 μM, RSL3 at 20 μM, shikonin at 5 μM or NaIO3 at 30 μM. Tissues were then collected 24 h later for flat mount or cryosection.
10
Expansion and Characterization of SCAP
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