MEF Rictor+/+ and MEF Rictor−/− cells were obtained from M. Magnuson at Vanderbilt University, Nashville, Tennessee (23 (link)). MAPK4-KO HCT116 cells were described previously (19 (link)). PNT1A cells were acquired from the European Collection of Authenticated Cell Cultures. Other cell lines were obtained from the American Type Culture Collection. MCF10A cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% horse serum (#16050122, Gibco), hydrocortisone (0.5 μg/ml; #H-0888, Sigma-Aldrich), human epidermal growth factor (20 ng/ml; #E9644, Sigma-Aldrich), insulin (10 μg/ml; #CAS11061-68-0, Santa Cruz Biotechnology), cholera toxin (100 ng/ml; #C-8052, Sigma-Aldrich), penicillin (100 U/ml), and streptomycin (100 μg/ml). MCF7 cells were cultured in DMEM with insulin (10 μg/ml); HCT116, PNT1A, PC3 cells in RPMI 1640; and other cell lines in DMEM, all supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml). Transient transfection and lentivirus packaging/infection were done as we previously reported (19 (link)).
E9644
Manufactured by Merck Group
Sourced in United States
The E9644 is a lab equipment product designed for general laboratory use. It serves as a core function in various experimental and analytical procedures. The detailed specifications and intended use of this product are not available for an unbiased and factual description.
Automatically generated - may contain errors
Lab products found in correlation
C8052, by Merck Group (4 mentions)
I9278, by Merck Group (4 mentions)
H0888, by Merck Group (3 mentions)
I0516, by Merck Group (3 mentions)
Epidermal growth factor (egf), by Merck Group (2 mentions)
S5261, by Merck Group (2 mentions)
Hydrocortisone, by Merck Group (2 mentions)
Phg0071, by Thermo Fisher Scientific (2 mentions)
Pnt1a, by European Collection of Authenticated Cell Cultures (1 mentions)
Heparin, by Merck Group (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Ascorbic acid a4403, by Merck Group (1 mentions)
F2293, by Merck Group (1 mentions)
D4902, by Merck Group (1 mentions)
Matrigel, by Corning (1 mentions)
F1141, by Merck Group (1 mentions)
C57bl 6j male mice, by Charles River Laboratories (1 mentions)
T8158, by Merck Group (1 mentions)
Lipofectamine 3000 transfection regent, by Thermo Fisher Scientific (1 mentions)
D1756, by Merck Group (1 mentions)
19 protocols using e9644
1
Cell Line Culture and Manipulation
2
Mammary Sphere Cell Culture Protocol
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Cells at 105 were washed with 1 × PBS and suspended in MEGM, including DMEM/F-12 (Invitrogen 11330), B27 (50x, Invitrogen 17504-044), Epidermal growth factor (EGF) (10 μg/ml, Sigma E9644; St. Louis, MO, USA), insulin (10 mg/ml, Sigma I9275), hydrocortisone (0.4 mg/ml, Sigma H0888), basic fibroblast growth factor (bFGF; 20 μg/ml, Sigma F0291), and heparin (8 mg/ml, Sigma). We seeded cells in a low attachment culture dish, then added MEGM to 10 ml, and incubated the mixture at 37 °C with 5% CO2. After a week, the number of sphere cells was counted under a microscope. Different views were randomly chosen, and averages were counted. Each sphere cell formation experiment was performed in triplicate.
3
Derivation of hc-iPSCs from Cumulus Cells
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The work is approved by Institutional Review Boards (IRB) at Mackay Memorial Hospital (MMH), IRB approval number: 14MMHIS261, and the Institutional Animal Care and Use Committee (IACUC) of National Taiwan University (NTU), IACUC approval number: NTU-103-EL-92. All the participants in this study understand the research and the written consent forms have been obtained from the patients. Patients were subjected to the controlled ovarian hyperstimulation (COH) treatments at MMH using the GnRH antagonist protocol as described [19 (link)]. Clinically discarded CCs were collected with patients’ consent. Basic information of the patients is shown in Table 1 . Freshly collected CCs from the same patient were pooled and immediately plated on matrigel (354234, Corning, NY, USA) coated 96-well dish in culture medium containing 2% fetal bovine serum (FBS, 16000–044), 0.05 mM ascorbic acid (A4403, Sigma-Aldrich, St. Louis, MO, USA), 0.05 μM dexamethasone (D4902, Sigma), 20 ng/mL epidermal growth factor (EGF, E9644, Sigma), basic fibroblast growth factor (bFGF, 13256–029, 50 ng/mL) and 1U/mL follicle stimulating hormone (FSH, F2293, Sigma). Medium was changed every 2–3 days after the cells attached on the dish. Twenty days post seeding, confluent cells were trypsinized by 0.05% Trypsin-EDTA. The cultured CCs at Passage 2 were subjected for hc-iPSC derivation.
4
Genetic Screening of E3 Ligase Knockdowns
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HeLa cells were transduced with three lentiviral shRNA pools containing 2,833 shRNAs against 616 E3 ligase genes or NS shRNA in triplicate at 0.2 multiplicity of infection (MOI) to prevent superinfection and to ensure that each cell received no more than one shRNA. After infection, HeLa cells were selected with puromycin (0.2 μg ml−1) for 7 days to enrich for HeLa cells expressing shRNA. After puromycin selection, HeLa cells were grown in serum-free DMEM containing trace elements (D0547, Sigma) supplemented with 50 ng ml−1 EGF (E9644, Sigma), 20 ng ml−1 FGF (SRP3043, Sigma), 100 nM hydrocortisone (H0888, Sigma), 0.5 μg ml−1 transferrin (T1147, Sigma), 5 ng ml−1 selenium (S5261, Sigma), 0.5 μg ml−1 fibronectin (F1141, Sigma) and 0.05 μg ml−1 insulin (I0516, Sigma). Media was changed every 3 days and cells were split at a 1:4 ratio every 7 days. After four passages all cells carrying NS and shRNAs from pool 2 were dead. Surviving cells from pools 1 and 3 were collected and genomic DNA was isolated. The integrated shRNAs were PCR-amplified using primers specific to the shRNA vector (pLKO.1) and listed in Supplementary Table 3 . Samples were sequenced using primer SP6 (Supplementary Table 3 ) to identify candidate shRNAs.
5
Culturing Rat Kidney Proximal Tubular Cells
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Rat kidney proximal tubular cells (RPTCs) were originally obtained from Dr Hopfer (Case Western Reserve University) and were cultured in DMEM/F12 medium (12400024; Gibco) containing transferrin (5 μg/mL) (T8158; Sigma), insulin (5 μg/mL) (I9278; Sigma), epidermal growth factor (EGF; 1 ng/mL) (E9644; Sigma), dexamethasone (4 μg/mL) (D1756; Sigma), 10% foetal bovine serum (FBS) (PAN‐Seratech) and 1% antibiotic‐antimycotic (15240062; Thermo Fisher Scientific). RPTC HNF1β knockdown and scramble cell lines were constructed in our previous study.17 Lipofectamine 3000 Transfection Regent (L3000008) was purchased from Thermo Fisher Scientific Company.
About 8‐week‐old C57BL/6J male mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd (China). Mice were acclimated for 7 days before formal experiments and were housed in groups of four animals per cage. Animal experiments were performed in accordance with the approval of the Ethics Committee for Experimental Animals in Henan University (Approval Number: DWLL20200102).
About 8‐week‐old C57BL/6J male mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd (China). Mice were acclimated for 7 days before formal experiments and were housed in groups of four animals per cage. Animal experiments were performed in accordance with the approval of the Ethics Committee for Experimental Animals in Henan University (Approval Number: DWLL20200102).
6
Oncosphere Formation and Radiation Response
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A total of 1 × 103 BrM cells were plated in low-attachment plates in Humec medium (12753018, Gibco) supplemented with 10 ng ml−1 basic human fibroblast growth factor (13256-029, Gibco), 20 ng ml−1 epidermal growth factor (EGF; E9644, Sigma-Aldrich), 5 µg ml−1 insulin solution from bovine pancreas (IGF1; I0516, Sigma-Aldrich), and B27 supplement (17500-044, Gibco). H2030-BrM cells were grown for 7 days and E0771-BrM cells for 4 days, so formed oncospheres were approximately the same size or number per well. After this period, oncospheres were irradiated with a single dose of 10 Gy; 72 h later, oncosphere size was evaluated using ImageJ.
7
Isolation of Primary Mouse Keratinocytes
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Primary mouse keratinocytes were isolated from epidermis of newborn control and Par3eKO mice. To separate epidermis from dermis, whole skins of P0–P3 mice were incubated in 5 mg/ml Dispase II (Sigma-Aldrich) diluted in DMEM/HAM’s F12 medium with low Ca2+ (50 μM) (Biochrom) supplemented with 10% FCS (chelated), penicillin (100 U/ml), streptomycin (100 μg/ml, Biochrom #A2212), adenine (1.8 × 10−4 M, SIGMA #A3159), l -glutamine (2 mM, Biochrom #K0282), hydrocortisone (0.5 μg/ml, Sigma #H4001), epidermal growth factor (10 ng/ml, Sigma #E9644), cholera enterotoxin (10−10 M, Sigma #C-8052), insulin (5 μg/ml, Sigma #I1882), and ascorbic acid (0.05 mg/ml, Sigma #A4034) at 4 °C overnight (termed FAD medium). After incubating the epidermis for 20 min in TrypLE (Gibco) at room temperature, dissociated cells were collected and cultured in low Ca2+ FAD medium on collagen-I-coated plates. Mixed litters were used to isolate control and Par3eKO cells. For cell culture experiments, cell–cell contact formation was induced by calcium switch, i.e. by supplementing the FAD medium with 1.8 mM CaCl2 for the indicated time points.
8
Cultivation of GFP-expressing Cancer Cells
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Human colorectal carcinoma cells HCT 116 (ATCC® CCL-247TM) stably expressing green fluorescent protein (GFP) [32 (link)] were cultured in Dulbecco’s Modified Eagle Medium DMEM + 4.5 g/L of glucose (Gibco-Invitrogen, Carlsbad, CA, USA), L-Glutamine (CSTGLU00, Eurobio, Les Ulis, France) and pyruvate, supplemented with 10% of fetal bovine serum (F7524, Sigma, Saint Louis, MI, USA) and 1% of penicillin/streptomycin (P0781, Sigma, Saint Louis, MI, USA). SKOV-3 stably expressing green fluorescent protein (GFP) and luciferase (Luc) ovarian carcinoma cells from (ATCC® HTB-77™) were cultured in RPMI 1640 (Eurobio Scientific), supplemented with L-Glutamine, 10% of fetal bovine serum, 1% of penicillin/streptomycin, human insulin (I9278, Sigma Aldrich, Saint-Louis, MI, USA) at 10 µg/mL and recombinant human epidermal growth factor (E9644 from Sigma Aldrich) at 20 ng/mL. Cells were kept in a humidified atmosphere at 37 °C and 5% of CO2 and were mycoplasma negative (as tested every week with MycoAlert Mycoplasma Detection kit, cat n°#LT07-318, Lonza, Switzerland).
9
Isogenic Cell Lines for Cancer Research
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MCF10A, a non-tumorigenic breast cell line (ATCC, CRL-10317) and MCF10A CDH1−/− cells (CLLS1042, Sigma-Aldrich, St Louis, MO, USA) were cultured in DMEM/F12/Glutamax (10565042, Thermo Fisher Scientific, Waltham, MA, USA) with 5% horse serum (16050, Thermo Fisher Scientific, Waltham, MA, USA), 10μg/μL neutral insulin (797139, Dunedin hospital, Dunedin, New Zealand), 500 ng/mL hydrocortisone (H0888, Sigma-Aldrich, St Louis, MO, USA), 100 ng/mL cholera toxin (C8052, Sigma-Aldrich, St Louis, MO, USA) and 20 ng/mL human epidermal growth factor (E9644, Sigma-Aldrich, St Louis, MO, USA). A CDH1-null strain of the gastric cancer cell line NCI-N87 (ATCC, CRL-5822) was generated using CRISPR-Cas9 [9 (link)]. Briefly, the sequence 5′-ATTCACATCCAGCACATCCA-3′ was used to target exon 10 of CDH1 and induce a single base frameshift, followed by a stop codon. Isogenic NCI-N87 cells were cultured in DMEM/F12/Glutamax with 10% fetal bovine serum (10091148, Thermo Fisher Scientific, Waltham, MA, USA). Both cell lines were grown at 37 °C with 5% CO2.
10
Culture of OSCC Cell Lines and Fibroblasts
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Isolated CAFs and NOFs from OSCC patients and healthy donors were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; D6429, SIGMA) supplemented with 10% heat-inactivated newborn calf serum (NBCS; 31765068, GIBCO). OSCC cell lines UK1 (29 (link)) and Luc4 (30 (link)) were grown in DMEM/Nutrient Mixture F-12 Ham medium (D8437, Sigma) supplemented with 10% NBCS, 1× Insulin-Transferrin-Selenium (41400-04, Thermofisher Scientific), 0.4 μg/ml hydrocortisone (H0888, Sigma), 50 μg/ml L-ascorbic acid (A7631, Sigma), and 10 ng/ml epidermal growth factor (E9644, Sigma). All cell lines were propagated in humidity incubator at 5% CO2 and 37°C temperature and regularly tested for mycoplasma contamination.
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