In vitro gene silencing assays were performed using primary mouse hepatocytes freshly isolated as previously reported (7 (link)). Free uptake and transfection assays were performed using protocols previously described (52 (link),53 (link)). Each siRNA concentration was analyzed in duplicate. For transfection assays, 5 μl siRNA at indicated concentrations were mixed with 4.9 μl of Opti-MEM (LifeTech, cat #31985–062) and 0.1 μl of Lipofectamine RNAiMax (Invitrogen, cat #13778150) per well of a 384-well plate. After incubation at room temperature for 15 min, 40 μl of William's E Medium (Life Tech, cat #A12176-01) containing ∼5×103 primary mouse hepatocytes were added to the wells. Cells were incubated for 24 h at 37°C in 5% CO2 prior to RNA purification. For free uptake experiments, 5 μl siRNA at indicated concentrations were mixed with 5 μl Opti-MEM (LifeTech, cat #31985-062) per well of a 384-well plate. After 15 min, 40 μl of William's E Medium (Life Tech, cat #A12176-01) or EMEM medium (ATCC) containing ∼5 × 103 cells were added to the siRNA mixture. Cells were incubated for 24 h at 37°C in 5% CO2 prior to RNA purification.
William s e medium
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany, France, Belgium, Switzerland, Japan, China, Italy
William's E medium is a cell culture medium designed for the growth and maintenance of a variety of cell types, including hepatocytes. It provides the necessary nutrients and growth factors to support cell proliferation and differentiation. The composition of William's E medium is optimized to support the specific requirements of hepatocyte cultures.
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Lab products found in correlation
L glutamine, by Thermo Fisher Scientific (30 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (29 mentions)
Insulin, by Merck Group (25 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (24 mentions)
Streptomycin, by Thermo Fisher Scientific (22 mentions)
Penicillin, by Thermo Fisher Scientific (20 mentions)
Glutamax, by Thermo Fisher Scientific (18 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (14 mentions)
Dexamethasone (dex), by Merck Group (14 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (13 mentions)
Hydrocortisone, by Merck Group (11 mentions)
Hepes, by Thermo Fisher Scientific (8 mentions)
Liver digest medium, by Thermo Fisher Scientific (7 mentions)
Hydrocortisone hemisuccinate, by Merck Group (7 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (7 mentions)
Stereomicroscope, by Olympus (7 mentions)
Streptomycin, by Merck Group (6 mentions)
Penicillin, by Merck Group (6 mentions)
Dexamethasone (dex), by Thermo Fisher Scientific (6 mentions)
L glutamine, by Merck Group (6 mentions)
265 protocols using william s e medium
1
Silencing Assays in Mouse Hepatocytes
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In vitro gene silencing assays were performed using primary mouse hepatocytes freshly isolated as previously reported (7 (link)). Free uptake and transfection assays were performed using protocols previously described (52 (link),53 (link)). Each siRNA concentration was analyzed in duplicate. For transfection assays, 5 μl siRNA at indicated concentrations were mixed with 4.9 μl of Opti-MEM (LifeTech, cat #31985–062) and 0.1 μl of Lipofectamine RNAiMax (Invitrogen, cat #13778150) per well of a 384-well plate. After incubation at room temperature for 15 min, 40 μl of William's E Medium (Life Tech, cat #A12176-01) containing ∼5×103 primary mouse hepatocytes were added to the wells. Cells were incubated for 24 h at 37°C in 5% CO2 prior to RNA purification. For free uptake experiments, 5 μl siRNA at indicated concentrations were mixed with 5 μl Opti-MEM (LifeTech, cat #31985-062) per well of a 384-well plate. After 15 min, 40 μl of William's E Medium (Life Tech, cat #A12176-01) or EMEM medium (ATCC) containing ∼5 × 103 cells were added to the siRNA mixture. Cells were incubated for 24 h at 37°C in 5% CO2 prior to RNA purification.
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In vitro gene silencing assays were performed using primary mouse hepatocytes freshly isolated as previously reported (7 (link)). Free uptake and transfection assays were performed using protocols previously described (52 (link),53 (link)). Each siRNA concentration was analyzed in duplicate. For transfection assays, 5 μl siRNA at indicated concentrations were mixed with 4.9 μl of Opti-MEM (LifeTech, cat #31985–062) and 0.1 μl of Lipofectamine RNAiMax (Invitrogen, cat #13778150) per well of a 384-well plate. After incubation at room temperature for 15 min, 40 μl of William's E Medium (Life Tech, cat #A12176-01) containing ∼5×103 primary mouse hepatocytes were added to the wells. Cells were incubated for 24 h at 37°C in 5% CO2 prior to RNA purification. For free uptake experiments, 5 μl siRNA at indicated concentrations were mixed with 5 μl Opti-MEM (LifeTech, cat #31985-062) per well of a 384-well plate. After 15 min, 40 μl of William's E Medium (Life Tech, cat #A12176-01) or EMEM medium (ATCC) containing ∼5 × 103 cells were added to the siRNA mixture. Cells were incubated for 24 h at 37°C in 5% CO2 prior to RNA purification.
2
Isolation and Culture of Chicken Primary Keratinocytes
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Chicken feet were disinfected with iodine and 70% ethanol. Skin fragments were excised and incubated with 0.5 mg/mL thermolysin (#T7902-100MG, Sigma) either 2 h at 37°C for 19-day old chickens or overnight at 4°C for older chickens. After its separation from the dermis, the epidermis was enzymatically digested 10 min at 37°C in a 0.05% trypsin-EDTA solution (#11580626, Gibco). Isolated chicken primary keratinocytes (CPKs) were filtered through a 40 μm mesh strainer (Falcon) and centrifuged 7 min at 1400 rpm. The CPKs pellet was resuspended in 3 mL William’s E medium (#32551–020, Gibco) supplemented with 3% chicken serum, 2% fetal calf serum, 1% L-glutamine and 1% penicillin-streptomycin (#DE17-602E, Lonza).
For the infectivity assay, 1 mL of the CPK suspension was added on two wells of a 6-well plate containing a confluent layer of CESCs. After an overnight co-culture, cells were washed with PBS and then cultivated 3 to 4 days with William’s E medium (#32551–020, Gibco) supplemented with 1.5% chicken serum, 1% fetal calf serum, 1% L-glutamine and 1% penicillin-streptomycin (#DE17-602E, Lonza).
For the infectivity assay, 1 mL of the CPK suspension was added on two wells of a 6-well plate containing a confluent layer of CESCs. After an overnight co-culture, cells were washed with PBS and then cultivated 3 to 4 days with William’s E medium (#32551–020, Gibco) supplemented with 1.5% chicken serum, 1% fetal calf serum, 1% L-glutamine and 1% penicillin-streptomycin (#DE17-602E, Lonza).
3
Hepatocyte Isolation and Insulin Stimulation
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Primary hepatocytes were isolated by collagenase/EGTA perfusion as published elsewhere (18 ) from male C3HeB/FeJ mice. Following preparation, hepatocytes were resuspended in Williams E Medium (Gibco, Paisley, UK, Catalogue Number 32551), 10% (v/v) FCS, 100 nm dexamethasone. The hepatocyte cell lines Hepa 1–6 and BNL CL.2 were obtained from ATCC (Manassas, VA) and cultured in DMEM (Gibco, Catalogue Number 41966), 10% (v/v) FCS.
For stimulation experiments, cells were seeded onto collagen I coated six-well plates and cultured in serum-free starvation medium (Williams E Medium and DMEM for primary hepatocytes and hepatocyte cell lines, respectively). Subsequently, cells were pre-treated with EETs (4 μm of 5(6)-EET, 8(9)-EET, 11(12)-EET and 14(15)-EET (Cayman Chemicals, Ann Arbor, MI) or vehicle (DMSO) in starvation medium for 1h at 37 °C 5% CO2. Next, cells were stimulated with starvation medium containing the indicated concentrations of human insulin (Sigma-Aldrich, Steinheim, Germany) with or without EETs at the same concentration as above. Stimulation media for this second step were supplemented with Phosphatase Inhibitor Mixture 2 and 3, (Sigma-Aldrich) at 1:200. DMSO was added to wells without EETs to yield the same final concentration in all wells of the experiment. Cells were incubated for 20, 40 or 60 min at 37 °C 5% CO2 and scraped into 50 μl RIPA buffer.
For stimulation experiments, cells were seeded onto collagen I coated six-well plates and cultured in serum-free starvation medium (Williams E Medium and DMEM for primary hepatocytes and hepatocyte cell lines, respectively). Subsequently, cells were pre-treated with EETs (4 μ
4
Maintenance and Culture of HeLa and Primary Hepatocytes
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HeLa cells were maintained in DMEM (Invitrogen) supplemented with 100U/ml penicillin and streptomycin (Invitrogen) and 10% fetal bovine serum (Gibco) at 37°C and 5% CO2. Human primary hepatocytes (Gibco) were thawed and plated in Williams E Medium (Gibco) supplemented with 5% fetal bovine serum, 1 μM dexamethasone in DMSO, 100 U/ml penicillin and streptomycin, 4 μg/ml human recombinant insulin, 2 mM GlutaMAX™, and 15 mM HEPES pH 7.4 at 37°C and 5% CO2. Plated hepatocytes were allowed to recover for 6 h and subsequently maintained in Williams E Medium (Gibco) supplemented with 0.1 μM dexamethasone in DMSO, 50 U/ml penicillin and streptomycin, 6.25 μg/ml human recombinant insulin, 6.25 μg/ml human transferrin, 6.25 μg/ml selenous acid, 6.25 μg/ml bovine serum albumin, 6.25 μg/ml linoleic acid, 2 mM GlutaMAX™,15 mM HEPES pH 7.4 and 5 mM CaCl2 at 37°C and 5% CO2.
5
Liver Spheroid Preparation Protocol
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Example 2
Preparation of Liver Spheroids
Liver spheroids are prepared as described in the GravityTRAP™ ULA Plate manual from InSphero. Briefly, HepaRG cells are first thawed at 37° C. for 2 minutes and then mixed into 9 ml of pre-warmed William's E medium (ThermoFisher Scientific, ref. 12551032) supplemented with Thaw, Plate & General Purpose supplement (ThermoFisher Scientific, ref. HPRG770) and GlutaMAX solution (ThermoFisher Scientific, ref. 35050061). Cells are then centrifuged for 2 minutes at 400×g before medium is replaced with fresh William's E medium with the same supplements as described above. About 5000 cells are then distributed per wells of a Corning® spheroid microplate (ref. 4520). 5 days later, medium is replaced with fresh William's E medium (ThermoFisher Scientific, ref. 12551032) supplemented with HepaRG Maintenance & Metabolism Supplement (ThermoFisher Scientific, ref. HPRG720) and GlutaMAX solution (ThermoFisher Scientific, ref. 35050061). One week later, spheroids are mature and ready to be used for the experiments.
6
Isolation and Culture of Mouse Hepatocytes
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Mouse hepatocyte primary cultures were established from the livers of adult Bsg+/+ and Bsg–/– mice according to the previously described method (44 (link)). In short, immediately following euthanasia of the animals, livers were perfused sequentially with buffered Liver Perfusion Medium (catalog 17701-038; Thermo Fisher Scientific) and Liver Digest Medium (catalog 17703-034; Thermo Fisher Scientific). Hepatocytes from the removed livers were extracted into William’s E Medium (catalog A1217601; Thermo Fisher Scientific) containing Primary Hepatocyte Thawing and Plating Supplements (catalog CM3000; Thermo Fisher Scientific). Primary cultured hepatocytes were maintained in William’s E Medium supplemented with Maintenance Supplements (catalog CM4000; Thermo Fisher Scientific).
7
Insulin signaling in BMP4-treated cells
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Cells were seeded on gelatin-coated plates and cultured in Williams E medium (Life Technologies, MA, USA), supplemented with 10% fetal bovine serum. For insulin signaling analysis, the cells were starved for 16 h with 0.1% serum and incubated with recombinant BMP4 (50 ng/mL), recombinant Gremlin1 (200 ng/mL), and/or insulin (10 nM), followed by protein and RNA extractions.
8
Cell Culture Protocols for Liver Cancer
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HepG2 cells were obtained from RIKEN BioResource Center (Tsukuba, Japan) and cultured in DMEM (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), L-glutamine, and 1% penicillin–streptomycin. JHH7 and JHH6 cells were purchased from the Japanese Cancer Research Resource Bank (Osaka, Japan) and cultured in Williams E medium (Life Technologies) supplemented with 10% FBS, L-glutamine, and 1% penicillin–streptomycin. These cell lines were maintained in a humidified atmosphere at 37°C and 5% CO2. Cell lines were authenticated in 2012 by cytogenesis.
9
Endometrial Epithelial-Stromal Co-culture
10
Culturing HepG2 and Primary Hepatocytes
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HepG2 cells were maintained in DMEM Glutamax supplemented with 10% v/v FBS, 50 U/ml penicillin and 50 µg/ml streptomycin, all provided by Life Technologies.
Primary mouse hepatocytes were isolated from adult mouse livers (ICR CD-1 mice, 8–14 weeks of age, Harlan Laboratories). The animals were anaesthetized with ketamin/xylazin and died during the perfusion procedure. The hepatocytes were isolated from the liver using a two-step collagenase perfusion method, followed by percoll gradient purification as described by Conçalves et al[13] (link). After isolation, cells were immediately seeded in micro-wells, or well plates coated with 0.1% collagen (BD Bioscienses), in order to compare three dimensional with two-dimensional cell culture.
Primary hepatocytes were cultured in William's E medium (Life Technologies), supplemented with L-glutamine (292 mg/ml) (Life Technologies), glucagon (7 ng/ml) (sigma), insulin (0.5 µg/ml) (sigma), hydrocortisone (25 µg/ml), EGF (10 ng/ml), 10% v/v FBS (Life Technologies), 50 U/ml penicillin (Life Technologies) and 50 µg/ml streptomycin (Life Technologies).
Both the HepG2 cells and the primary hepatocytes were maintained in a humidified 5% CO2-containing atmosphere at 37°C.
Primary mouse hepatocytes were isolated from adult mouse livers (ICR CD-1 mice, 8–14 weeks of age, Harlan Laboratories). The animals were anaesthetized with ketamin/xylazin and died during the perfusion procedure. The hepatocytes were isolated from the liver using a two-step collagenase perfusion method, followed by percoll gradient purification as described by Conçalves et al[13] (link). After isolation, cells were immediately seeded in micro-wells, or well plates coated with 0.1% collagen (BD Bioscienses), in order to compare three dimensional with two-dimensional cell culture.
Primary hepatocytes were cultured in William's E medium (Life Technologies), supplemented with L-glutamine (292 mg/ml) (Life Technologies), glucagon (7 ng/ml) (sigma), insulin (0.5 µg/ml) (sigma), hydrocortisone (25 µg/ml), EGF (10 ng/ml), 10% v/v FBS (Life Technologies), 50 U/ml penicillin (Life Technologies) and 50 µg/ml streptomycin (Life Technologies).
Both the HepG2 cells and the primary hepatocytes were maintained in a humidified 5% CO2-containing atmosphere at 37°C.
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