The HNSCC cell lines HLA-A2-EGFR+ PCI-15B and JHU-029 (Disis et al., 2009 (link); Lopez-Albaitero et al., 2009a (link); Andrade Filho et al., 2010 (link)), were grown in Iscove’s modified Dulbecco’s medium (IMDM; Sigma) supplemented with 10% FBS (Cellgro), 2% L -glutamine, and 1% penicillin/streptomycin (Invitrogen) at 37°C in a 5% CO2, 95% humidity. Adherent tumor cells were detached by warm Trypsin–EDTA (0.25%) solution (Invitrogen).
Trypsin edta 0.25 solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
Trypsin-EDTA (0.25%) solution is a cell detachment reagent used to dissociate adherent cells in cell culture applications. It contains 0.25% trypsin and 0.1% EDTA in Hank's Balanced Salt Solution.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Corning (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (2 mentions)
Ficoll paque plus, by Cytiva (1 mentions)
Iscove modified dulbecco media (imdm), by Merck Group (1 mentions)
Easysep kits, by STEMCELL (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Atcc crl 1435, by American Type Culture Collection (1 mentions)
Humid incubator, by Panasonic (1 mentions)
Antibiotic antimycotic solution, by Wisent (1 mentions)
Phosphate buffered saline (pbs), by Solarbio (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Transwell insert, by Corning (1 mentions)
Alamar blue solution, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Fluorobrite dmem, by Thermo Fisher Scientific (1 mentions)
14 protocols using trypsin edta 0.25 solution
1
HNSCC Cell Line Cultivation Protocol
2
Isolation and Characterization of NK Cells and HNC Cell Lines
3
In Vitro Cytotoxicity Evaluation of Extracts
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Human epidermal HaCaT keratinocytes were obtained from ATCC® and maintained in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin (10,000 U mL−1), and grown as monolayer at 37 °C in a 5% CO2 atmosphere. After three days the culture was approximately 80% confluent, cells being harvested with trypsin-EDTA 0.25% solution (GIBCO, Invitrogen, Grand Island, NY, USA). Then the cells were subcultured in 96-well plates (1.5 × 104 cells well−1), grown until 80% confluence, and subsequently exposed to the extract for 24 h at 37 °C. Interference with the mitochondrial activity was studied 24 h later by the MTT reduction assay as described in Gomes et al. [50 (link)]. Viable cells were calculated as a percentage of the negative control cells set at 100%, results corresponding to the mean ± SEM of three independent experiments performed in triplicate.
4
Bone Marrow Cell Culture Protocol
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Bone marrow cells were obtained from the tibiae and femurs by flushing and were cultured in Dulbecco's modified Eagle's medium (DMEM; ThermoFisher Scientific, Waltham, MA, USA), 10% fetal bovine serum (ThermoFisher Scientific), and 1% penicillin/streptomycin (ThermoFisher Scientific) in a humidified incubator at 37°C with 5% atmospheric CO2. The medium was changed every 2-3 days and, when the culture reached 90% confluency, the cells were passaged with trypsin-EDTA 0.25% solution (ThermoFisher Scientific).
5
Isolation and Expansion of Murine MSCs
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Bone marrow-derived MSCs were obtained from GFP transgenic C57Bl/6 male mice aged 4-8 weeks. The animals were kept in the animal facility of the Center for Biotechnology and Cell Therapy of Hospital São Rafael (Salvador, Brazil), with access to food and water ad libitum. The procedures for obtaining the cells were approved by the ethics committee for animal use of Hospital São Rafael (CEUA-HSR 007/18). Bone marrow cells obtained from the tibiae and femurs by flushing were centrifuged at 300 × g for 10 min. The pellet was resuspended in 10 ml of Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (all Thermo Fisher Scientific, Waltham, MA, United States) and cultured in plastic flasks in incubator at 37°C with 5% atmospheric CO2. After two days, the culture medium was completely changed and the non-adherent cells were removed. The adhered cells were cultured at 37°C with 5% atmospheric CO2 and the medium was changed every 3 days until the monolayer reached 90% confluence. The MSCs were detached using a trypsin-EDTA 0.25% solution (Thermo Fisher Scientific) for expansion and use for transduction.
6
Culturing mCRPC Cell Line PC3
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The mCRPC cell line PC3 (ATCC® CRL-1435™) was purchased from the American Type Culture Collection (ATCC) and cultured at 37°C and 5% CO 2 in a humid incubator (Panasonic, Japan). The Roswell Park Memorial Institute (RPMI)-1640 medium w/ sodium pyruvate and L-glutamine (Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, USA), and 1% antibiotic-antimycotic solution (Wisent Bio Products, USA) was used as a growth medium and changed every 2 days. Cells were passaged with a Trypsin-EDTA 0.25% solution (Thermo Fisher Scientific, USA) as needed.
7
Synthesis and Evaluation of mPEG-Coupled Doxorubicin Nanoparticles
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a-Methoxy-3-amino poly (ethylene glycol) (mPEG-NH2, Mn = 2 kDa) was purchased from Ponsure Biotech (Shanghai, China). 1-Chloro-3-hydroxypropane (Macklin, China), DL-glutamic acid (Macklin, China), 2-nitroimidazole (Accela, China), and doxorubicin hydrochloride (Melone Pharma, China) were used as received. A dialysis bag (Mw cutoff: 3.5 kDa) was purchased from Shanghai Green Bird Technology Development Co., Ltd. (Shanghai, China). Anhydrous N, N-dimethylformamide (DMF) was purchased from JK chemical (Beijing, China). Menthol, diethyl ether, petroleum ether, and ethyl acetate were purchased from Zhengzhou Yinfeng Reagent Co., Ltd. (Zhengzhou, China). Petroleum ether and ethyl acetate were dried over CaH2 and then distilled under ambient pressure.
MCF-7 cells were obtained from the Kunming cell library of the Chinese Academy of Sciences. MEM medium was purchased from HyClone (America). Phosphate-buffered saline (PBS) and 1% penicillin–streptomycin double antibody were purchased from Solarbio (Shanghai, China). Fetal bovine serum (FBS) was purchased from SeraPro (Germany). Trypsin–EDTA (0.25%) solution was purchased from Gibco (America). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo.
MCF-7 cells were obtained from the Kunming cell library of the Chinese Academy of Sciences. MEM medium was purchased from HyClone (America). Phosphate-buffered saline (PBS) and 1% penicillin–streptomycin double antibody were purchased from Solarbio (Shanghai, China). Fetal bovine serum (FBS) was purchased from SeraPro (Germany). Trypsin–EDTA (0.25%) solution was purchased from Gibco (America). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo.
8
Caco-2 Cell Surface BCRP Expression Analysis
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Caco-2 cells were seeded on 12mm, 0.4 μm Transwell® inserts (Corning®) at a density of , and incubated at 37°C, 5% CO2 for 21 d. During this period, cells were cultured in 10% FBS and 1 antimycotic-antibiotic supplemented DMEM, which was replaced with fresh media every 2–3 d. After 21 d, Caco-2 monolayer was washed with 1 PBS, and cells were treated with trypsin-EDTA 0.25% solution (Gibco™) for 20 min, and centrifuged at 300 RCF for 5 min. Harvested cells were washed in ice cold FACS buffer (1 PBS, 0.5% BSA, 0.1% NaN3), and their numbers were adjusted to . Next, Caco-2 cells were labeled with a PE-conjugated BCRP (ABCG2) monoclonal antibody (Invitrogen, Catalog # 12-8888-42) following a FACS cell surface staining protocol (M edicine.yale, 2020 ).
All FACS analysis were performed using the Thermo Fisher Attune™ NxT Flow Cytometer (Cornell Biotechnology Resource Center, Flow Cytometry Facility).
All FACS analysis were performed using the Thermo Fisher Attune™ NxT Flow Cytometer (Cornell Biotechnology Resource Center, Flow Cytometry Facility).
9
Abalone Extract Cytotoxicity Assay
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HaCaT cells were trypsinized from subconfluent cultures by adding Trypsin-EDTA (0.25%) solution (Gibco BRL), and incubated for 10 min with regular gentle shaking. The trypsin reaction was stopped by adding 10 ml of DMEM (Gibco BRL) containing 10% FBS. The cell suspension was then centrifuged at 1,000 × g for 5 min at 25°C. The cell pellet was re-suspended in 2 ml of culturing medium with 5% FBS and mixed by vortexing. The cells were counted with a hemocytometer. Then, 1×105 cells were equally seeded into each well of a 96-well black plate and incubated at 37°C for 24 h. After that, the culturing medium was removed and replaced with 200 μl of serial abalone extracts in 1% serum-media. Serial concentrations of abalone extracts were from 0.1 to 50 mg/ml. The cells were then incubated at 37°C for 24 h and 100 μl of a 10% AlamarBlue solution (Invitrogen, Carlsbad, CA, USA) in serum-free medium was directly added to each well. The plate was incubated at 37°C for 4 h and the absorbance was measured at 540 and 630 nm with a fluorescent spectrophotometer (Epoch; BioTek, Winooski, VT, USA). The percentage of cell viability was analyzed by GraphPad Prism version 6 (GraphPad Software, Inc., San Diego, CA, USA) using one-way ANOVA at P<0.001.
10
Isolation of Gallbladder Epithelial Cells
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When sacrificed, mice were anesthetized using phenobarbital. Upon opening the abdomen, the gallbladder was exposed and cystic duct was ligated. The gallbladder was soon removed avoiding liver tissues at the gallbladder bed. After aspiration of the bile, the gallbladder was washed with D-Hanks solution. Thereafter, the gallbladder was cut into pieces and put in 1.5 ml Trypsin-EDTA (0.25%) solution (No. 25200-056, Gibco®) and incubated at 37°C for 20 min and then centrifuged at 1500 rpm for 5 min. After removing the supernatant, the pellet was suspended in Trypsin-EDTA solution and repeated the digestion process two more times. The collected cell pellets were then suspended in Gibco® RPMI 1640 medium. The living singles cells were then picked by a mouth pipette and transferred to cold scRNA-seq lysis buffer (Vieira Braga and Miragaia, 2019 (link); Denisenko et al., 2020 (link)). To minimize experimental variation, all the procedures were performed by one expertise researcher.
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