Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza (Walkersville, MD, USA) and grown at 37°C and 5% CO2 in EGM™ BulletKit™ medium (Lonza, CC‐3162: EBM2 + FBS, Hydrocortisone, hFGF, VEGF, R3‐IGF, Ascorbic acid, hEGF, Heparin, GA‐1000) according to the manufacturer's recommendation. The HUVECs were passaged by trypsinization (0.25%, Corning, 25‐053‐CI, NY, USA), and only passages 3–5 were used for experiments. Human melanoma MDA‐MB‐435 and human embryonic kidney 293T (HEK 293T) cell lines were grown in DMEM (Corning, 10‐013‐CVR); human TERT‐Retinal Pigment Epithelium 1 (RPE1) cells were cultured in DMEM F‐12 (Corning, 10‐090‐CVR); and human ductal breast epithelial tumor cell line T47D and mouse primary lung epithelial tumor cell line TC‐1 were maintained in RPMI 1640 (corning, 15‐040‐CVR) at 37°C and 5% CO2. All of cell culture media were supplemented with 10% fetal bovine serum (FBS) (Corning, 35‐016‐CV) and 1% penicillin–streptomycin (PS) (Corning, 30‐002‐CI).
Trypsinization
Manufactured by Corning
Sourced in United States
Trypsinization is a laboratory technique used to detach adherent cells from a culture surface. Trypsin, a proteolytic enzyme, is used to cleave the cell-surface proteins that anchor the cells to the substrate. This process allows for the harvesting and passage of cell cultures.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Nd7 23, by Merck Group (2 mentions)
Dulbecco s phosphate buffered saline dpbs, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Corning (2 mentions)
Fluorolog fluorometer, by Horiba (2 mentions)
6 well plate, by Thermo Fisher Scientific (2 mentions)
Egm bulletkit medium, by Lonza (1 mentions)
Penicillin streptomycin p s, by Corning (1 mentions)
Huvecs, by Corning (1 mentions)
Dulbecco modified eagle medium (dmem), by Corning (1 mentions)
Dmem f12, by Corning (1 mentions)
Rpmi 1640, by Corning (1 mentions)
Vinblastine, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Acetic acid, by Merck Group (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Cytexpert, by Beckman Coulter (1 mentions)
Phosphate buffered saline (pbs), by MP Biomedicals (1 mentions)
γ secretase inhibitor compound e, by Merck Group (1 mentions)
9 protocols using trypsinization
1
Culturing Human Cell Lines for Experimental Studies
2
NGF-Induced Neuronal Differentiation
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The neuronal hybridoma cell line ND7/23 (Sigma Aldrich, St. Louis, MO) was maintained in Dulbecco’s modified Eagle’s medium (DMEM) (D6046; Sigma Aldrich) with 10% [vol/vol] of fetal bovine serum (FBS; Sigma Aldrich) + 1% [vol/vol] penicillin/streptomycin (Corning Inc., Corning, NY). Cells were multiplied until passage 10 (freshly thawed cells were considered passage 1). They were passaged by trypsinization (Corning Inc.) every 3–5 days, when approximately 70–90% confluent. Cells were seeded at a density of 5x104/cm2. After attachment, they were washed twice with 1x Dulbecco’s phosphate-buffered saline (dPBS; Thermo Fisher Scientific, Waltham, MA) and with serum free medium (SFM) and then incubated with SFM for 1 day to inhibit mitosis before adding nerve growth factor (NGF) [46 (link), 47 (link)]. Such SFM-primed cells were used in all experiments below, unless otherwise specified.
3
Preparation and Counting of Murine Metaphase Chromosomes
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LβT2 metaphase specimens were prepared by the Mouse Genetics and Gene Targeting CoRE at the Icahn School of Medicine at Mount Sinai (ISMMS) according to standard cytogenetic procedures for cultured cells [24 ]. Briefly, cell division was blocked at the metaphase stage by adding the spindle poison vinblastine (#V1377, Sigma) for 3 hours. Following trypsinization (#25-052-Cl, Corning, Corning, NY), cells were incubated in a hypotonic solution for 15 minutes (which makes the cell swell, thus allowing easy rupture of the cell membrane) and preserved in a swollen state with Carnoy’s fixative solution (methanol/glacial acetic acid 3:1; methanol, #650609, Sigma; acetic acid, #A6283, Sigma). Chromosome spreads were prepared by dropping fixed cell suspensions from a height onto cold slides, completely drying the slides, and staining them in a Giemsa-staining solution (#89002, Thermo Fisher Scientific, Waltham, MA). Chromosome counting was done manually using an inverted microscope at 600× magnification by visualizing Giemsa-stained chromosome spreads on a monitor. Plastic was overlaid on the monitor, and chromosomes were marked one by one with a Sharpie; after the marks were wiped clean, chromosomes in the next cell were counted. Chromosomes were counted in at least 20 cells.
4
Annexin V Apoptosis Assay by Flow Cytometry
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Following treatment, cells were collected by trypsinization (Corning), washed three times with phosphate buffer saline (PBS, Corning), and stained for Annexin V, using an Annexin V-FITC apoptosis detection kit (BD PharMingen), for 45 min, at room temperature, as previously described50 (link). The percentage of Annexin V-positive cells was determined by flow cytometry on a CytoFlex, using the CytExpert (v1.2, both Beckman Coulter). Reported are average values with standard errors, in a population of 10,000 cells, from three independent experiments. The original source images for a representative data and Annexin V gating strategies can be found in the Supplementary Fig. 12 .
5
Evaluating γ-secretase Inhibitor Effects on HPV16 Infection
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γ-secretase inhibitor (Compound E, referred to as XXI in this manuscript) was purchased from EMD Millipore (Billerica, MA) and dissolved in DMSO (Sigma-Aldrich, St. Louis, MO). We started testing different concentrations of XXI, 200 nM-3.2 μM (dilution factor 1:2), and 1 nM-250 nM (dilution factor 1:3), and 1 pM-900 pM (dilution factor 1:3). To test the effect of different concentrations of XXI on HPV16 PsV infection, we incubated cells on ice for 1 hour to slow down endocytosis, and prepared the DMEM-10 containing either XXI in DMSO or DMSO as control. After 1 hour of ice incubation, the media was washed off and DMEM-10 containing either XXI or DMSO was added to the wells. Following this, the cells were infected with HPV16 PsV and kept on ice for another 2 hours. After the ice incubation, the plates were directly put in the 37°C incubator for 48 hours. The cells were then collected via trypsinization (Corning, Manassas, VA) and washed three times with PBS (MP biomedicals, Solon, OH) by spinning at 3000 x g for 5 min. Percentage of infected HaCaT cells was calculated based on the number of GFP positive cells as measured by flow cytometer.
6
Cell Culture Protocols for Cancer and Stem Cell Research
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LN229, U251 (human glioblastoma), MDA-MB-231 (human breast cancer), HEK-293 (human embryonic kidney), and Hela cell lines were cultured in Dulbecco’s Modified Eagle Media (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) (WISENT), 1% penicillin and streptomycin (Gibco) and passaged by trypsinization (Corning®). SH-Sy-5y (human neuroblastoma) cell line was purchased from ATCC and passaged in DMEM-F12 supplemented with 10% FBS, 1% MEM Non-Essential Amino Acids Solution (NEAA) (Gibco), 1% penicillin and streptomycin (Gibco). Low-passage GSC (GBM4 and GBM8) cells were provided by Dr. Hiroaki Wakimoto (MGH) and cultured as neurospheres in Neurobasal medium (Gibco) supplemented with 0.5% N-2 (Gibco), 2% B-27 (Gibco), 1.5% glutamine (Gibco), 0.02 ng/ml epidermal growth factor (EGF) (Gibco), and 20 ng/ml fibroblast growth factor (FGF) (PeproTech) in ultralow attachment flasks or plates. GSC neurospheres were passaged using the NeuroCult chemical dissociation kit (Stemcell Technologies). Human astrocytes were cultured in astrocyte medium (ScienCell Research Laboratories) supplemented with 2% FBS, 1% antibiotic solution and 1% astrocyte growth supplement (ScienCell Research Laboratories) and passaged using cell/tissue dissociation solution (Innovation Cell Technologies, Inc.). All cells were maintained at 37 °C and 5% CO2.
7
ND7/23 Cell Line Culture and Priming
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The neuronal hybridoma cell line ND7/23 (Sigma Aldrich, St. Louis, MO) was maintained in Dulbecco's modified Eagle's medium (DMEM) (D6046; Sigma Aldrich) with 10% [vol/vol] of fetal bovine serum (FBS; Sigma Aldrich) + 1% [vol/vol] penicillin/streptomycin (Corning Inc., Corning, NY). Cells were multiplied until passage 10 (freshly thawed cells were considered passage 1). They were passaged by trypsinization (Corning Inc.) every 3-5 days, when approximately 70-90% confluent. Cells were seeded at a density of 5x10 4 /cm 2 . After attachment, they were washed twice with 1x Dulbecco's phosphate-buffered saline (dPBS; Thermo Fisher Scientific, Waltham, MA) and with serum free medium (SFM) and then incubated with SFM for 1 day to inhibit mitosis before adding nerve growth factor (NGF) [46, (link)47] (link). Such SFM-primed cells were used in all experiments below, unless otherwise specified.
8
Fluorescence Spectra of Biosensors
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Emission spectra of biosensors were obtained using a Fluorolog fluorometer (Horiba). HEK-293t cells grown in 6-well plates (Nunc) were transfected with biosensor DNA plus regulator if required. After 24 h cells were detached by trypsinization (Cellgro) and resuspended in cold PBS (Sigma) + 1%FBS (Hyclone), washed and then resuspended in cold PBS. Samples were excited at 430nm and spectra obtained from 460 to 600nm for biosensors with Cerulean3/TagCFP/mTFP, and 550 to 650nm for LSSmOrange biosensors. Dual chain biosensors were corrected for acceptor bleedthrough.
9
Fluorescence Spectra of Biosensors
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