HepG2 cells were seeded using a Multiflo FX multi-mode automated reagent dispenser (Biotek, UK) at 2.0 × 10 5 cells per well (2D) in Ibidi microplate 96 well black plates (Ibidi, USA) and incubated for 24 h at 37 °C in a Forma Steri-Cycle CO 2 incubator (Thermo Scientific, UK) or 400 cells per well (3D) in 96-well ULA round-bottomed plates (Corning, UK) in 180μL of complete DMEM. Cells were incubated for 4, 11 and 21 days 37°C in a Forma Steri-Cycle CO 2 incubator (Thermo Scientific, UK). Post-seeding CYP1A2 and CYP2E1 mRNA expression in HepG2 2D and 3D cultures were measured relying on RT-PCR. The results are expressed as percent mean mRNA content normalised to GAPDH and are the mean ± SD of n = 3 experiments.
Forma steri cycle co2 incubator
Manufactured by Thermo Fisher Scientific
Sourced in United States, Singapore, United Kingdom
The Thermo Scientific Forma Steri-Cycle CO2 Incubator is a laboratory equipment designed for cell culture applications. The incubator provides a controlled environment for the growth and maintenance of cell cultures, with precise temperature, humidity, and carbon dioxide (CO2) concentration regulation.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Mem alpha modification, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Low melt agarose, by Bio-Rad (2 mentions)
Phenol red, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Szx12, by Olympus (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
Hyclone, by GE Healthcare (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Cardiomyocyte characterization kit, by Merck Group (1 mentions)
Penicillin streptomycin, by Lonza (1 mentions)
L glutamine, by Lonza (1 mentions)
Ccl 75tm, by American Type Culture Collection (1 mentions)
Sv30010, by Thermo Fisher Scientific (1 mentions)
Actinred 555, by Thermo Fisher Scientific (1 mentions)
Mesencult basal media, by STEMCELL (1 mentions)
27 protocols using forma steri cycle co2 incubator
1
Quantifying CYP1A2 and CYP2E1 in 2D and 3D HepG2 Cells
2
Anchorage-Independent Colony Formation Assay
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Trypsinized cells were resuspended in RPMI-1640 media without phenol red (#R8755, Sigma) containing 0.35% low-melt agarose (#1613111, Bio-Rad, Hercules, CA, USA) and 10% FBS (Sigma). 2,000 cells suspended in agarose-containing media were plated on top of a 0.5% agarose base layer. The cells were cultured in a Forma Steri-Cycle CO2 incubator (Thermo Fisher) CO2 incubator at 37° C with 5% CO2 for 3 weeks and stained with 0.05% crystal violet (Gentian Violet, ICM Pharma, Singapore). For image capture of colonies, a dissection microscope (SZX-12, Olympus, Tokyo, Japan) was used while the colony numbers and sizes were quantitated using the Image J software [63 (link)].
3
MSC Cultivation and Expansion Protocol
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The collected MSCs from different tissue origins were separately seeded into 75 cm2 cell culture flask at the density of 1 × 106/cm2 with DMEM/F12 (11320033, GIBCO) medium plus 10% FBS, 2 mM L-glutamine and 1% penicillin/streptomycin solution. Cells were cultivated at 37 °C in a saturated humidified atmosphere containing 5% CO2 in a Forma Steri‑Cycle CO2 Incubator (Thermo Fisher Scientific, Inc.). After 72 h, the non-adherent cells were removed by washing with PBS solution, after then the medium was changed every three days. The MSCs cultured to the density of 80% following treatment with 0.05% trypsin (27250018, GIBCO) and 0.02% EDTA (AM9912, Invitrogen) for 3 min at 37 ˚C. The cells were washed by centrifugation at 350 × g for 5 min, then replanting to a new T75 cell culture flask at a lower density. MSCs were cultured to the third passage (P3) and prepared for the next step experiment.
4
Culturing Human Fetal Lung Fibroblasts
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Human foetal lung fibroblasts (WI-38, ATCC, CCL-75TM) were maintained in HyClone minimum essential medium (MEM) Alpha Modification (Thermo Scientific, Waltham, MA, USA) containing 10% (v/v) foetal bovine serum (FBS) (Gibco/Invitrogen, Burlington, ON, Canada) in a humidified Forma Steri-Cycle CO2 Incubator (Thermo Scientific) at 37°C and 6% CO2. The population doubling (PD) number of a given passage was determined by summing up the ΔPD = log2(nf/ni) for each subculture, where nf is the final number of cells in a culture and ni is the number of cells initially inoculated.
5
Clonogenic Assay for Cell Survival
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Trypsinized cells were resuspended in RPMI-1640 media without phenol red (#R8755, Sigma–Aldrich) containing 0.35% low-melt agarose (#1613111, Bio-Rad) and 10% FBS (Sigma). The cell suspension was plated on top of a 0.5% agarose base layer. The cells were allowed to grow in a Forma Steri-Cycle CO2 incubator (Thermo Fisher) CO2 incubator at 37°C with 5% CO2 for 3 weeks and stained with 0.05% crystal violet (Gentian Violet, ICM Pharma, Singapore). Images of colonies were acquired using a dissecting microscope (SZX-12, Olympus, Tokyo, Japan) and the colony numbers and sizes were quantitated using Image J software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, imagej.nih.gov/ij/ , 1997–2016).
6
Isolation and Expansion of UC-MSCs
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UC-MSCs were isolated and expanded from human umbilical cords as described previously (1 (link)). UC-MSCs were cultured in Dulbecco's modified Eagle medium (DMEM) with nutrient mixture F-12 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal calf serum (FCS; HyClone; GE Healthcare Life Sciences, Logan, UT, USA), 100 U/ml penicillin (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany), 100 µg/ml streptomycin (Sigma-Aldrich; Merck Millipore), 2 mM glutamine and 10 ng/ml epidermal growth factor (PeproTech, Inc., Rocky Hill, NJ, USA). This mixture medium is referred to as culture medium. Cells were cultured at 37°C in a humidified atmosphere containing 5% carbon dioxide in a Forma™ Steri-Cycle™ CO2 Incubator (Thermo Fisher Scientific, Inc.). UC-MSCs were stained with rhodamine phalloidin and DAPI and observed under a confocal microscope.
7
Cardiomyocyte Differentiation and Characterization
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Cells were extracted as described in [18 (link)], in which collagenase digestion of murine left ventricles was followed by centrifugal separation. Cultures were prepared in Cardiomyocyte Differentiation Medium (StemCell Technologies) and seeded on two scaffolds: One on a nanofiber scaffold treated with a thymosin β4 coating; the other was prepared on an uncoated scaffold for comparison. Stem cells were seeded at a concentration of 1×106 over both scaffolds and allowed to grow in a cultured medium. Cells were incubated in a Forma Steri-Cycle CO2 Incubator (Thermo Scientific, Waltham, MA) at 36.5°C and 3.0% CO2. Cardiomyocytes were characterized based on control and antibody staining as described in the Cardiomyocyte Characterization Kit protocol (Millipore).
8
Senescence in Human Fetal Lung Fibroblasts
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Human foetal lung fibroblasts (WI-38, ATCC, CCL-75TM) were maintained in HyClone minimum essential medium (MEM) Alpha Modification (ThermoScientific) containing 10% (v/v) foetal bovine serum (FBS) (Gibco) in a humidified Forma Steri-Cycle CO2 Incubator (ThermoScientific) at 37°C and 6% CO2. In order to compare the senescence-associated changes, three different population doubling (PD) levels of cells were used: PD 38 (proliferatively active), PD 47 (pre-senescent) and PD 54 (senescent), as described previously (manuscript 000141R1 in AGING).
9
Immortalized Murine Microglia Culture
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Spontaneously immortalized murine microglia, SIM-A9_CRL-3265 (American Type Culture Collection, ATCC, Manassas, VA, USA) cells were cultured as previously described [21 (link)]. Briefly cells were cultured in DMEM/F-12 medium_DFL15 (Caisson Laboratories, Smithfield, UT, USA) containing 10% FBS, 5% HS, and 1% P/S. Next, cells were gently shaken and detached from the culture vessel with phosphate buffered saline (PBS) containing 1 mM EDTA, 1 mM EGTA and 1 mg/mL D-glucose. The detached cells were counted with a hemocytometer, and rested for at least 24 h in a Forma Steri-Cycle CO2 incubator (Thermo Scientific, Waltham, MA, USA) at 37 °C, 95% humidity and 5% CO2, before treatments. The cell line can yield more than 5 million cells after culture and was used for western blotting experiments (Figure 1 , Figure 2 and Figure 3 ).
10
Culture and Subculture of NCI-H69V Cells
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The NCI‐H69V cells (purchased from the European Collection of Authenticated Cell Cultures [ECACC], Cat# 91091803, Public Health England, UK), was cultured using standard culturing conditions. The culture medium, Roswell Park Memorial Institute (RPMI 1640) medium (Gibco, Thermo Fisher Scientific, Johannesburg, South Africa) was supplemented with 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific), 1% non‐essential amino acids (NEAA) (Lonza; Whitehead Scientific [Pty] Ltd.), 1% penicillin/streptomycin (10,000 U of each/ml, Lonza; Whitehead Scientific [Pty] Ltd.) and 2 mM L‐glutamine (Lonza; Whitehead Scientific [Pty] Ltd.). Cultures were incubated in a Forma Steri‐Cycle CO2 Incubator (Thermo Fisher Scientific; Marietta) at 37°C with 5% CO2 and 95% humidified air, with the culture medium exchanged every second day. Sub‐culturing of the cells was initiated upon reaching 80%–90% confluence by trypsinization, using 0.25% Trypsin‐Versene.
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