Heracell 150i

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany, China, Australia, Japan, France, Denmark

The Heracell 150i is a CO2 incubator designed for cell and tissue culture applications. It features a stainless steel interior and is equipped with a Thermo Scientific infrared CO2 sensor for precise CO2 control.

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Heracell™ 150i Tri-Gas Incubator HERAcell 150i or 160i 5% CO2 incubator HERAcell 150i/240i Heracell 150i incubator Heracell 150i CO2 incubator Heracell

140 protocols using heracell 150i

HepG2 cell culture protocol

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Maintenance of HepG2 cells was done in DMEM medium having 10% FBS and 1x antibiotic-antimycotic solution. Plating of cells was done in 96 well-plates or 60 mm or 100 mm at the density of 1 × 10⁴ cells/mL or 1 × 10⁶ or 3 × 10⁶ plates, respectively for 24 h followed by treatment. Culturing of treated and untreated cells was done in normoxia incubator (Heracell 150i, Thermo Scientific, USA) maintaining 4% CO₂ at 37°C.
HepG2 cells were maintained in DMEM medium containing FBS (10%) and 1x antibiotic-antimycotic solution. Cells were plated in a density of 1 × 10⁴ cells/mL in 96 well-plates or 1 × 10⁶ cells/mL in 60 mm plates or 3 × 10⁶ cells/mL in 100 mm plates and maintained for 24 h following treatments. Treated and untreated cells were cultured in normoxia incubator (Heracell 150i, Thermo Scientific, USA) maintaining 5% CO₂ at 37°C in DMEM medium.

Chester K., Paliwal S., Khan W, & Ahmad S. (2017). UPLC-ESI-MS/MS and HPTLC Method for Quantitative Estimation of Cytotoxic Glycosides and Aglycone in Bioactivity Guided Fractions of Solanum nigrum L.. Frontiers in Pharmacology, 8, 434.

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Cytotoxicity of TCEP in HepG2 cells

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MTT and NRU assays were performed to quantify the cytotoxicity of tris(2-chloroethyl) phosphate (TCEP) (purity 97%, Cat. No. 119660, CAS No. 115-96-8, Sigma-Aldrich, St. Louis, MO, USA) in HepG2 cells. The HepG2 cell line was obtained from American Type Culture Collection (ATCC) (Beltsville, MD, USA). Selection of TCEP exposure concentrations and duration of exposure were first analyzed in pilot experiments by treating the HepG2 cells for 1 to 3 days in an incomplete RPMI-1640 medium supplemented with low (5, 10, 25, 50 μM) and high (100, 200, 400 μM) concentrations of TCEP in a CO₂ incubator (5%, 95% humidity) at 37 °C (Hera Cell 150i, Thermo Scientific, Langenselbold, Germany). No cytotoxic effects were found at the above-mentioned concentrations after 2 days of exposure (data not shown). Only the higher concentrations (100, 200, 400 μM) of TCEP showed significant cytotoxicity after 3 days. Consequently, these concentrations were selected for further experiments.

M. Al-Salem A., Saquib Q., Siddiqui M.A., Ahmad J, & Al-Khedhairy A.A. (2020). Tris(2-chloroethyl) Phosphate (TCEP) Elicits Hepatotoxicity by Activating Human Cancer Pathway Genes in HepG2 Cells. Toxics, 8(4), 109.

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Adhesion of Functionalized Microbubbles on HUVECs

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HUVECs were cultured in the supplemented endothelial cell growth
medium at 37 °C in a 5% CO₂/95% air humidified atmosphere. Flow
chamber slides (μ-Slides I 0.4, Ibidi) were coated with 2% gelatin
solution then 2.5 × 10⁵ HUVECs were seeded. The μ-Slides
were put upside down in the incubator (HeraCell 150i, Thermo Fisher
Scientific) and left in this position overnight to allow cells to
grow and adhere on the roof of the chamber slides. Afterward, cells
were washed three times with PBS and placed in an upright position
under an inverted microscope (Nikon Inverted Microscope Eclipse Ti-E,
Florence, Italy) equipped with a 40× objective (Nikon, Japan),
a motorized stage, and the Zyla sCMOS camera 4.2 (Andor, Belfast,
U.K.) for video recording. Nontargeted plain bubbles and c-RGDfC functionalized lipid MBs (10⁶ MBs/mL) were pumped
into the channel slide with a syringe pump at a constant flux of 0.76
mL/min, corresponding to a shear stress of 1 dyn/cm². After
10 min, five different fields of view were captured using bright field
microscopy (Nikon NIS-Element AR, Florence, Italy). Then, the channel
slide was inverted upside down for 5 min, placed upright under the
microscope, and perfused further for 10 min with PBS to wash out the
unspecifically adhered and easily detachable MBs. Pictures corresponding
to five different fields of view were captured again.

Oddo L., Paradossi G., Cerroni B., Ben-Harush C., Ariel E., Di Meco F., Ram Z, & Grossman R. (2019). In Vivo Biodistribution of Engineered Lipid Microbubbles in Rodents. ACS Omega, 4(8), 13371-13381.

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Polypropylene Tube Cell Isolation Protocol

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In this work, various materials were used. These materials are 50 polypropylene conical tubes (Baxter, Toronto, ON, Canada); Sterile cell strainer: 40 μm nylon mesh (Millipore, Burlington, MA, USA); 11 μm nylon filters (Millipore, Burlington, MA, USA); 1.5 mL Eppendorf tubes (Fisherscience, Saint-Laurent, QC, Canada); 0.9% irrigation-grade sodium chloride solution (Baxter, Toronto, ON, Canada). We also employed several instruments, including an incubator (Heracell 150i, Thermo Fisher Scientific, Waltham, MA, USA); Lab Centrifuge (Sorvall ST 8, Thermo Scientific, Waltham, MA, USA); Hemocytometer (BLAUBRAND^® Neubauer Millipore Sigma, Burlington, MA, USA); Inverted and phase contract Microscope (isherbrand^TM Inverted Infinity, Phase contrast 10× and 20×, light splitter (100% or 20/80%), Fisher Scientific, Hampton, NH, USA).

Osouli Tabrizi H., Panahi A., Forouhi S., Sadighbayan D., Soheili F., Haji Hosseini Khani M.R., Magierowski S, & Ghafar-Zadeh E. (2022). Oral Cells-On-Chip: Design, Modeling and Experimental Results. Bioengineering, 9(5), 218.

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Detailed Cell Culture Protocol

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The cell lines used were HeLa (ATCC^® CCL-2™) and a murine DCs line (DC 2.4 [40 (link)], an immortalized murine bone marrow derived DCs line), and were grown according to the typical culture procedure detailed here. The cell culture medium for DCs was composed of RPMI-1640, heat-inactivated Fetal Bovine Serum (FBS) (10%), 2-mercaptoethanol (50 µM) and HEPES buffer solution (10 mM). HeLa cells were cultured in DMEM containing 10% heat-inactivated FBS (all reagents were purchased from Gibco, Ireland and Life Technologies, Carlsbad, CA, USA). For both cell lines, after aspirating the old culture medium, the cells that adhered to the bottom of the flask T75 were washed twice with 10 mL of PBS. Then, 1 mL of trypsin solution (0.25% trypsin-EDTA) was added to the cells and let for 3–10 min at 37 °C. Then, the trypsin solution containing the cells was mixed with 9 mL of fresh complete culture medium. After centrifugation (5 min at 300× g), the appropriate amount of cells was resuspended in 13 mL of fresh culture medium in a new T75 flask, and cultured in a 37 °C incubator (Heracell 150i, ThermoFisher Scientific, USA) under 5% CO₂ and 95% humidity. Cells were used with a low passage number (less than 10).

Lamrayah M., Phelip C., Coiffier C., Lacroix C., Willemin T., Trimaille T, & Verrier B. (2022). A Polylactide-Based Micellar Adjuvant Improves the Intensity and Quality of Immune Response. Pharmaceutics, 14(1), 107.

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Hypoxia and Irradiation Protocol

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Hypoxic conditions were obtained in a tri-gas chamber under an atmosphere containing 1% O₂, 5% CO₂ and 94% N₂ at 37 °C (Heracell 150i; Thermo Fisher Scientific, Waltham, MA, USA, or 9000E; Wakenyaku, Kyoto, Japan). During chronic hypoxia, cells were maintained in hypoxic conditions over several passages before experiments, whereas acute hypoxia corresponded with 0–24 h of hypoxia. During irradiation, flasks with a non-filtered cap were closed and then opened when they were replaced in the incubator. Additionally, during trypsinization, the culture medium was enriched with nitrogen to limit the reoxygenation of the cells.

Wozny A.S., Vares G., Alphonse G., Lauret A., Monini C., Magné N., Cuerq C., Fujimori A., Monboisse J.C., Beuve M., Nakajima T, & Rodriguez-Lafrasse C. (2019). ROS Production and Distribution: A New Paradigm to Explain the Differential Effects of X-ray and Carbon Ion Irradiation on Cancer Stem Cell Migration and Invasion. Cancers, 11(4), 468.

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Overexpression of PLAGL2 in SW480 Cells

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SW480, human colon adenocarcinoma cell line, was obtained from the Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Science (SIBS; Shanghai, China) and cultured in Dulbeccos modified Eagles medium(DMEM) supplemented with 10% fetal bovine serum (FBS) and kept in a humidified incubator (Heracell 150i; Thermo Fisher Scientific, Langenselbold, Germany) at 37°C, 5% CO₂. After reaching 90–95% confluency, SW480 cells were transfected with the vector pcDNA3.1- PLAGL2 using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturers instructions. Forty-eight hours later, G418, 200 mg/l (Invitrogen) was added to the culture medium for cell selection. Single or mixed clonal populations of stably transfected cells were grown under G418 selection for 1–2 weeks and followed by routine DMEM culture for 2 weeks. The expression level of PLAGL2 in G418-resistant clones was thereafter evaluated by real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis.

Wang Y.P., Guo P.T., Zhu Z., Zhang H., Xu Y., Chen Y.Z., Liu F, & Ma S.P. (2017). Pleomorphic adenoma gene like-2 induces epithelial-mesenchymal transition via Wnt/β-catenin signaling pathway in human colorectal adenocarcinoma. Oncology Reports, 37(4), 1961-1970.

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Isolation of Palatal Shelf Epithelial Cells

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Mouse embryonic heads at E16.5 were collected from both groups (normal group (n = 3) vs. model group (n = 3)). The samples were fixed in liquid nitrogen for 5 min and stored at −80 °C for 7 days. The samples were sent to Guangzhou Fitgene Biotechnology Co., Ltd. (Guangdong, China) on dry ice to isolate and culture the palatal shelf epithelial cells. The cells were cultured in Minimum Essential Medium (Hyclone, Logan, UT, USA) containing 10% fetal bovine serum (Hyclone) in a 5% CO₂ and 37 °C incubator (Heracell 150i; Thermo Fisher Scientific, Waltham, MA, USA). Cells from passage 3 were used for subsequent experiments.

Zhang M., Zhou J., Ji Y., Shu S., Zhang M, & Liang Y. (2023). LncRNA-NONMMUT100923.1 regulates mouse embryonic palatal shelf adhesion by sponging miR-200a-3p to modulate medial epithelial cell desmosome junction during palatogenesis. Heliyon, 9(5), e16329.

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Canine Lymphoma Cells Under Hypoxia

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Canine lymphoma cells (CL-1 and GL-1) [14 (link), 15 (link)], DOX-resistant lymphoma cells (CL-1DR and GL-1DR) and mononuclear cells were used in this study. The CL-1DR and GL-1DR were generated from the corresponding parental cells (CL-1 and GL-1) with a previously reported procedure [16 (link)], the details of which are given in the S1 File and S1 Table. Mononuclear cells were isolated from the fresh peripheral blood of a healthy 1-year-old, intact female beagle by a specific gravity centrifugal method using LymphoPrep (Cosmo Bio, Tokyo, Japan). All cells were cultured in RPMI 1640 (Gibco, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Cosmo Bio, Tokyo, Japan) and 1% L-glutamine (BioWhittaker, Walkersville, MD, USA) under various O₂ concentrations (21%, 10%, 5%, or 1% O₂) with 5% CO₂ at 37°C in a tri-gas incubator (HERAcell® 150i; Thermo Scientific, Waltham, MA, USA).

Yamazaki H., Lai Y.C., Tateno M., Setoguchi A., Goto-Koshino Y., Endo Y., Nakaichi M., Tsujimoto H, & Miura N. (2017). Hypoxia-activated prodrug TH-302 decreased survival rate of canine lymphoma cells under hypoxic condition. PLoS ONE, 12(5), e0177305.

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Cultivation of MCF-7 Breast Cancer Cells

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MCF-7 cells, a breast cancer cell line from an invasive breast ductal carcinoma, were cultivated in medium consisting of RPMI 1640, 10% fetal bovine serum and 1% penicillin streptomycin solution. The cells were maintained at 37 °C and 5% CO₂ in a Heracell 150i (Thermofisher Scientific™) incubator in a 75 cm² cell culture flask. The culture was split every three to four days using a trypsin/EDTA solution (Sigma-Aldrich).

Wimmenauer C, & Heinzel T. (2023). Identification of nanoparticles as vesicular cargo via Airy scanning fluorescence microscopy and spatial statistics. Nanoscale Advances, 5(13), 3512-3520.

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