MCF-7 cell line purchased from NCCS, Pune was cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin solution. The cell line was maintained in a water-jacketed CO2 incubator (Forma series 2, Thermo Fisher, India) at 37 °C and 5% CO2. To ensure culture was free from mycoplasma contamination, cells were tested and authenticated. The cells were sub-cultured routinely, every three to four days [19 (link)].
Forma series 2
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Forma Series II is a line of laboratory equipment designed for controlled temperature and environmental conditions. The core function of this product is to provide a stable and consistent environment for various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal calf serum (fcs), by Atlanta Biologicals (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
35 mm glass bottom culture dishes, by MatTek (1 mentions)
P50g 1.5 14 f, by MatTek (1 mentions)
Af 100 15, by Thermo Fisher Scientific (1 mentions)
Matrigel, by Corning (1 mentions)
Matrigel, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Heracell 150, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Euroclone (1 mentions)
Lncap, by American Type Culture Collection (1 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Euroclone (1 mentions)
Rpmi 1640, by Euroclone (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Solarbio (1 mentions)
Caco 2, by American Type Culture Collection (1 mentions)
Hyclone, by GE Healthcare (1 mentions)
14 protocols using forma series 2
1
Culturing MCF-7 Breast Cancer Cells
2
Cytotoxicity of T. indica and C. fistula Gums
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The cytotoxicity of crude and carboxymethylated gums from T. indica and C. fistula seeds was determined by MTT assay of exposed Caco-2 cells. This assay measures the conversion of MTT to insoluble formazan by dehydrogenase enzymes of the intact mitochondria of living cells. Caco-2 cells (passage 8–28) were seeded at a density of 104 cells/well with 100 µl of the culture medium into 96-well plates. After 48 h incubation at 37 °C in a CO2 incubator (Thermo Forma Series 2, Ohio, USA), the culture medium was replaced and the confluent cells were incubated for 4 h with 0, 0.01, 0.025, 0.05, 0.075, and 0.1 µg/ml of the carboxymethylated gums dispersed in PBS. After treatment, each carboxymethylated gum sample was removed and fresh medium was added before incubating the cells for 4 h to stabilize the cells. Finally, the cells were incubated with MTT diluted in medium (0.1 mg/ml) for 4 h. The medium was removed and the formazan crystal that formed in the living cells was dissolved in 100 µl DMSO per well. The relative viability (%) was calculated based on absorbance at 550 nm using a microplate reader. Viability was determined by comparing absorbance in wells containing treated cells with that of untreated cells. Data were obtained from three independent experiments, with three replicates per treatment point.
3
Bioglass Foam Scaffold Aging Protocol
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Scaffolds with an average porosity of 63% and pore size distribution within the 50–750 µm range were machined into cylinders (9 mm in diameter) and manually cut with a circular diamond saw into discs of 6 mm in thickness. The porosity was evaluated according to Equation (1):
where = 2.7 g cm−3 is the theoretical density of 45S5 Bioglass® [20 ].
To assess the effect of aging on foam properties, the samples were subjected to accelerated aging treatments in an incubator (Forma Series II, Thermo Electron Corporation, Waltham, MA, USA) at 37 °C, 90% relative humidity (RH), and 5% CO2 for up to 8 weeks. Ten samples for each aging time point were used. The control (pristine) specimens were sealed and maintained under vacuum to be used as reference samples, while the remaining ones were placed in open glass bottles and put in the incubator for aging. After aging, the samples were dried in an oven (ISOTEMP 550D, Fisher Scientific, Hampton, NH, USA) at 100 °C for 24 h to remove residual absorbed water.
where = 2.7 g cm−3 is the theoretical density of 45S5 Bioglass® [20 ].
To assess the effect of aging on foam properties, the samples were subjected to accelerated aging treatments in an incubator (Forma Series II, Thermo Electron Corporation, Waltham, MA, USA) at 37 °C, 90% relative humidity (RH), and 5% CO2 for up to 8 weeks. Ten samples for each aging time point were used. The control (pristine) specimens were sealed and maintained under vacuum to be used as reference samples, while the remaining ones were placed in open glass bottles and put in the incubator for aging. After aging, the samples were dried in an oven (ISOTEMP 550D, Fisher Scientific, Hampton, NH, USA) at 100 °C for 24 h to remove residual absorbed water.
4
HepG2 Cell Culture Protocol
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EXAMPLE 15
Cell Culture: HepG2 cells were grown in Eagle's Minimum Essential Medium (EMEM) (Invitrogen, Grand Island, N.Y., USA) supplemented with 10% fetal calf serum (Atlanta Biologicals, Lawrenceville, Ga., USA). Penicillin/streptomycin (1%) was also present in the culture media (Invitrogen). The cells were trypsinized and collected by centrifugation, and then the cell pellet was resuspended in suitable media. An aliquot (1 mL) of the cell suspension was transferred to 35-mm glass-bottom culture dishes (MatTek Corp., Ashlan, Mass., USA) and the cells was allowed to incubate for 24 hours at 37° C. in a 5% CO2 atmosphere (Thermo Electron Corp., Forma Series II).
5
CXCL12 Release Kinetics from Alg/Chit Nanoparticles
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The remaining Alg/Chit NPs pellet was resuspended into sterile phosphate buffer saline (PBS) (350 µL) in order to mimic physiological conditions and transferred to low binding microcentrifuge tubes to avoid non-specific adsorption. These tubes were then incubated in a humidified cell-culture incubator (Forma Series II, water jacket CO2 incubator, Thermo Electron Corporation, Gormley, ON, USA) at 37 °C for different time points between 0 and 168 h under static conditions. After each time point (0; 0.5; 1; 2; 4; 6; 8; 24; 48; 72; 168 h), tubes were centrifuged at 20,000× g (10 °C) for 30 min. Supernatants were collected and the fluorescence was quantified. The amount of CXCl12-AF647 released was determined as a percentage of cumulative mass release, as described by the following equation:
where Mt represents the total mass of released CXCL12-AF647 at time “t”, Mi the cumulative mass of solute released at time t and Minitial the initial loaded mass determined experimentally.
The pellet was then resuspended again with fresh PBS and put back to the incubator for the next sampling. After 168 h of incubation, Alg/Chit NPs were dissolved into Tris (10 mM)-EDTA (1 mM) buffer for 20 min at room temperature and the remaining concentration of CXCL12-AF647 was determined by fluorescence quantification.
where Mt represents the total mass of released CXCL12-AF647 at time “t”, Mi the cumulative mass of solute released at time t and Minitial the initial loaded mass determined experimentally.
The pellet was then resuspended again with fresh PBS and put back to the incubator for the next sampling. After 168 h of incubation, Alg/Chit NPs were dissolved into Tris (10 mM)-EDTA (1 mM) buffer for 20 min at room temperature and the remaining concentration of CXCL12-AF647 was determined by fluorescence quantification.
6
Cell Culture and Exosome Isolation
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HEK-293T, Beas-2B, A549, SPC-A1, SPC-A1-BM cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; HyClone, SH30243.01) while H520, H1581, HCC95, and H2170 cells were cultured in Roswell Park Memorial Institute (RPMI; HyClone, SH30809.01) containing 10% fetal bovine serum (FBS; LONSA, S711-001S) and 1% penicillin-streptomycin (PS; HyClone, SV30010). Cells were incubated at 37°C in a 5% CO2 incubator (Thermo, Forma Series II) for follow-up experiments. SPC-A1-BM is a highly bone metastatic cell line established from SPC-A1 by in vivo selection in BALB/c mouse models (Yang et al., 2009 (link); Yu et al., 2014 (link); He et al., 2019 (link)).
To deplete bovine exosomes from FBS, FBS was diluted to 20% by conditioned medium and then centrifuged at 120,000 ×g for 16 h at 4°C. For exosomes isolation, cultures were incubated with exosome-free FBS for 24 h prior to collecting the cell culture medium.
To deplete bovine exosomes from FBS, FBS was diluted to 20% by conditioned medium and then centrifuged at 120,000 ×g for 16 h at 4°C. For exosomes isolation, cultures were incubated with exosome-free FBS for 24 h prior to collecting the cell culture medium.
7
Organoid Culture in Matrigel Microenvironment
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About 900 epithelial clusters (1.0 gland equivalent) with a 20,000–50,000 stromal/mesenchymal cell addition (0.2 gland equivalent) were embedded in Matrigel (Corning #356234) at a 1:1 cell to Matrigel ratio. Ten microliters of the cell and basement membrane mixture was seeded into the well of a 50 mm glass-bottom dish (MatTek #P50G-1.5-14F). The Matrigel was solidified by incubating at 37 °C in a tissue culture incubator (Thermofisher Scientific Forma Series II) for 15 min and covered with 180 µL of DMEM/F-12/10% FBS/Pen-Strep with or without growth factors added. The growth factor concentrations used were 100 ng/ml epidermal growth factor (EGF) (PeproTech #AF100-15) or fibroblast growth factor-2 (FGF2) (Peprotech #450-33) solubilized in 0.2% BSA and stored at −20 °C in single-use aliquots. Organoids were cultured for 7 days at 37 °C in a tissue culture incubator in 5% CO2 with the medium replaced once at day 4. After 7 days of culturing, organoids were fixed by replacing the medium with 4% paraformaldehyde (PFA) (Electron Microscopy Sciences #15710) in 1× PBS for 20 min and stored in 1× PBS prior to Raman imaging or immunocytochemistry.
8
Maintenance of HEK293T and Jurkat Cells
9
Isolation and Culture of Human Placental Mesenchymal Stem Cells
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Placentas were harvested from donors at The First Affiliated Hospital, School of Medicine, Zhejiang University. All protocols for handling human tissues and cells were approved by the Ethics Committee of The First Affiliated Hospital of Zhejiang University (No. 2013-272). The isolation and culture of hP-MSCs was performed as described previously[16 (link),17 (link)]. The cells were stabilized in a standard humidified incubator (HERAcell150, Thermo Fisher Scientific Inc., Waltham, MA, United States) with a 21% O2 and 5% CO2 atmosphere. The hypoxic groups were placed in a humidified, water-jacketed CO2 incubator with oxygen control (Forma™ Series II, Thermo Fisher Scientific Inc.) in an atmosphere containing 2.5% O2 and 5% CO2. The normoxia group continued to be incubated in the standard humidified incubator.
10
Hypoxic Culture of Prostate Cancer Cells
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The androgen-dependent human prostate cancer cell line, LNCaP, and the androgen-independent cell line, PC-3, were obtained from ATCC (LGC Standards S.r.l., Italy), maintained in liquid nitrogen and used within few weeks after thawing and plating. Cells were grown in RPMI-1640 (EuroClone) (LNCaP) and DMEM (PC-3), supplemented with 10 % (vol/vol) fetal bovine serum (FBS) (EuroClone), L-glutamine (EuroClone) and 1 % (vol/vol) antibiotic/antimycotic solution (Gibco, Invitrogen S.r.l.). Hypoxia was achieved by maintaining the cells at 2 % oxygen, for 24 h, in a CO2 incubator (Forma Series II, Thermo Scientific) with oxygen sensor control, and CO2 and N2 gas regulators [18 (link)].
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