Hippocampi were dissected from E19 Sprague-Dawley male and female rat pups (Charles River, Wilmington, MA) in ice-cold HEPES-buffered saline solution (Gibco, Gaithersburg, MD), supplemented with penicillin-streptomycin (Lifetech, Guangdong, China), glucose, and additional HEPES. Hippocampi were dissociated using papain (Worthington Biochemical, Lakewood, NJ). Cells were washed and resuspended in phenol-red-free Neurobasal Medium (Lifetech) supplemented with L-glutamine (Stem Cell Technologies, Vancouver, Canada) and SM1 (NeuroCult; Stem Cell Technologies). Mixed neuron and astrocyte cell suspension was plated at a concentration of 200,000 cells/mL onto glass bottom 35 mm petri dishes (CellVis, Mountain View, CA) coated with poly-L-lysine (Sigma-Aldrich, St. Louis, MO). Cells were incubated in a humidified 37°C, CO2 incubator (HeraCell, Thermo Fisher Scientific, Waltham, MA). At DIV (days in vitro) 20, cells were transfected with GCaMP6f using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) for 48 h before imaging.
Heracell
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The Heracell is a laboratory incubator designed for cell and tissue culture applications. It provides a controlled environment for the growth and maintenance of cell lines and other biological samples. The Heracell incubator offers precise temperature, humidity, and gas concentration regulation to support optimal cell growth conditions.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Mycoprobe mycoplasma detection kit, by R&D Systems (3 mentions)
Rpmi 1640, by Corning (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Papain, by Worthington (1 mentions)
L glutamine, by STEMCELL (1 mentions)
Tcs sp5, by Leica camera (1 mentions)
Hepes, by Lonza (1 mentions)
Ampflstr identifier plus pcr amplification kit, by Thermo Fisher Scientific (1 mentions)
3500 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Mcf 7, by Thermo Fisher Scientific (1 mentions)
Bt 549, by Thermo Fisher Scientific (1 mentions)
Dld 1, by Thermo Fisher Scientific (1 mentions)
Af 100 15, by Thermo Fisher Scientific (1 mentions)
Streptomycin p s, by Thermo Fisher Scientific (1 mentions)
Hek293t cells, by Thermo Fisher Scientific (1 mentions)
Glutamax 1, by Thermo Fisher Scientific (1 mentions)
24 protocols using heracell
1
Culturing Rat Hippocampal Neurons for Calcium Imaging
2
Culturing Immortalized Keratinocytes on Hydrogels
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Human skin immortalized keratinocytes (Hacats) have been widely used to study psoriasis and related lesions [40 (link)]. They were cultured on the Rb-SA/PAAm gels. Each group has four parallel samples. The steps were followed as described in previous studies [41 ,42 (link)]. In short, the Rb-SA/PAAm gel was sterilized with ethyl alcohol (75%), before those cells were inoculated. After being soaked in dulbecco's modified eagle medium (DMEM), the hydrogels were expanded to equilibrium state. The cells on the hydrogels were stained with Thermo Scientific HERAcell (150i, U.S.A.) and then photographed by laser scanning confocal microscope using (TCSSP5, Leica, Germany). As described in previous work, CCK8 method was used to quantify the cell growth on hydrogels [43 (link)].
3
Culturing Immortalized Liver Cell Lines
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The hepatoma cell lines and the non-neoplastic, SV40 large T antigen-immortalized, hepatocyte lines, PH5CH1, PH5CH7, and PH5CH8 [22 (link)] were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (AQMedia, Sigma-Aldrich) supplemented with 10 % fetal calf serum (PAA), 100 mg/l streptomycin, 60 mg/l penicillin, and 25 mM HEPES (Lonza). The SV40 large T antigen-immortalized human liver epithelial cells (THLE-2) were cultured in LHC-8 medium supplemented with 70 ng/ml phosphoethanolamine, 5 ng/ml epidermal growth factor, 10 % FBS, 100 mg/l streptomycin, and 60 mg/l penicillin. Cells were grown at 37 °C in a water-saturated atmosphere at 5 % CO2. Hypoxia treatment was performed at 37 °C in a water-saturated atmosphere at 5 % CO2 in a hypoxia chamber (C-Chamber (C-274 & C-374) with a ProOx C21 Static O2 & CO2 Controller from BioSpherix) at the indicated oxygen percentage. HIF-1 α and HIF-2 α screening experiments were performed in an hypoxia incubator (Heracell) from Thermo Scientific at 37 °C in a water-saturated atmosphere at 5 % CO2. Human oncostatin M (227 a.a.) was from PeproTech. For all experiments, cells were seeded together, stimulated for the indicated periods of time, and harvested together at the latest time point.
4
TNBC Cell Line Characterization and Validation
5
Cytotoxicity Evaluation of Cip-MS-G-TSG
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The cytotoxicity of Cip-MS-G-TSG was employed on HaCaT cell lines, obtained from Chinese Academy of Sciences. Cells were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin and 100 μg/mL streptomycin at 37 °C in humidified incubator (HERAcell, ThermoFisher, America) with 95% air and 5% CO2. Cells were seeded in a 96-well plate at a density of 5 × 103 per well and incubated for 24 h. Afterwards, the growth medium was incubated with different concentrations of Cip-MS-G-TSG or PBS solution in DMEM containing 10% FBS. After 24 and 48 h incubation, the cell viability was characterized by Thiazolyl Blue Tetrazolium Bromide (MTT) colorimetric assays. The optical absorbance of each well (n = 6) was measured at 490 nm using a Microplate Absorbance Reader (ELX800, BioTek Instruments, America).
6
Cell Line Characterization and Maintenance
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SUM149 (RRID: CVCL_3422) cell lines were obtained from Prof. Zhiming Shao from the Fudan University Shanghai Cancer Center and maintained with F12 medium with 5% FBS, Insulin (5 μg/ml) and Hydrocortisone (1 μg/ml). Other cell lines, including MDA-MB-231 (RRID: CVCL_0062), T47D (RRID: CVCL_0553), MCF7 (RRID: CVCL_0031), BT-549 (RRID: CVCL_1092), DLD1 (RRID: CVCL_0248), A549 (RRID: CVCL_0023) and H460 (RRID: CVCL_0459) were obtained from ATCC. All cell lines were regularly tested for mycoplasma contamination using the Mycoprobe mycoplasma detection kit (R and D Systems, Minneapolis, MN). MCF-10A (RRID: CVCL_0598) cells were maintained with DMEM/F12 medium with 5% horse serum, Insulin (10 μg/ml), EGF ((PeproTech, Cat# AF-100-15, 20 ng/ml), Cholera Toxin (100 ng/ml) and Hydrocortisone (0.5 μg/ml). The other cells were maintained with growth media (DMEM for MDA-MB-231, MCF7, DLD1 and A549, and RPMI 1640 for BT-549, H460 and T47D) cultured in Heracell (Thermofisher, Waltham, MA) humidified incubators at 37 °C and 5% CO2.
7
Culturing Human and Mouse Cell Lines
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Human embryonic kidney (HEK) 293T cells (ATCC, #CRL-3216) and NIH 3T3 cells (ATCC, #CRL-1658) were cultured in Dulbecco's Modified Eagle Media (DMEM, Invitrogen #10313021) supplemented with 1% GlutaMax I and 100 unit/ml penicillin and 100 μg/ml streptomycin (P.S., all reagents from Life Technologies) and 10% fetal bovine serum (FBS, not heat-inactivated, HyClone, #SH30071.03, Thermo Scientific) under normoxia (20% O2, 5% CO2, at 37°C) using HERAcell (Thermo Scientific). Human patient skin fibroblasts (Congenital heart disease patient, #CDH01–0634) were cultured in DMEM supplemented with 1% GlutaMax I, P.S. and 20% FBS under normoxia (20% O2, 5% CO2, at 37°C) using HERAcell. The patient fibroblasts were obtained by taking a small piece of skin at the incision at the time of surgery following a protocol approved by the Columbia Institutional Review Board (IRB, for W.K. Chung) (23 (link)). Mouse embryonic fibroblasts (MEF) were isolated from E12.5 mouse embryos. MEF were cultured in DMEM supplemented with 1% GlutaMax I, P.S and 10% FBS under normoxia (20% O2, 5% CO2, at 37°C) using HERAcell. Human fibroblasts, MEF and NIH3T3 cells were passaged using 0.25% trypsin + 0.03% ethylenediaminetetraacetic acid (EDTA) while HEK 293T cells were passaged using 0.05% trypsin + 0.03% EDTA solution (both from Gibco/Life Technologies).
8
Cultivation of PC12 and hPheo1 Pheochromocytoma Cells
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Rat pheochromocytoma cell line, PC12 [13 (link)], and the human progenitor pheochromocytoma cell line, hPheo1 [14 (link)], were generous gifts from Arthur Tischler and Hans K. Ghayee. PC12 cells were cultivated in RPMI-1640 containing 10% horse serum (HS) and 5% fetal calf serum (FCS) on collagen A-coated cell culture dishes. For hPheo1 cells, RPMI-1640, containing 10% FCS and 2 mM Glutamax, was used (detailed experimental procedures and results are summarized in Supplementary Materials ). If not stated otherwise, cells were cultivated under normoxic conditions (5% CO2, 37 °C, and 95% humidity) in a CO2 incubator (HERAcell, Thermo Scientific). For the simulation of hypoxic conditions, cells were incubated in reduced oxygen partial pressure (≤1% O2) using an incubator equipped with an oxygen-sensor (Gasboy, Labotect, Göttingen, Germany). All cells were passaged up to 10 times after thawing, and experiments were conducted after passaging the cells at least once. Therefore, cells were trypsinized (trypsin/EDTA; 0.05%/0.02%), diluted with complete medium and counted by using C-CHIPs (Neubauer improved). All cell lines and the subsequently described sub-cell lines were regularly tested to be mycoplasma free (MycoAlert Mycoplasma Detection Kit, Lonza, Basel, Switzerland).
9
Cell Culture Conditions for Cancer Cell Lines
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A549 lung cancer, MDA-MB-231 breast cancer and B16F10 mouse melanoma cell lines were obtained from National Centre for Cell Sciences (NCCS), Pune, India and were cultured in DMEM (PAN BIOTECH GmbH, Aidenbach,Germany) with 10% fetal bovine serum (FBS), supplemented with 1% L-glutamine and 1% penicillin - streptomycin (Himedia Laboratories Pvt. Ltd., Mumbai, India). The maintenance of cells was done at 37 °C in a humidified, 5% CO2 atmosphere in an incubator (Hera Cell, Thermo Scientific,Waltham, MA).
10
ICSI Oocyte Denudation and Fertilization
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Cumular cells were removed from the oocytes with a denudation pipette (1–2 h following oocyte sampling by ovum pick up) using 80 IU/ml hyaluronidase (in Sydney IVF Fertilization medium) for 10–15 sec. Following the partial removal of cumular cells, the oocytes were further denudated (Sydney IVF Fertilization medium) until complete denudation. The denudated oocytes were placed in a cultivation box (Heracell; Thermo Fisher Scientific, Waltham, MA, USA) under conditions of 37°C, 5% O2, 6% CO2 and 89% N2, until required for ICSI. The sperm were injected into the oocytes 2–3 h following ovum pick up. Oocytes used for ICSI were in metaphase II (MII) after extrusion of the first polar body.
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