Mouse 3T3-L1 preadipocytes (#CL-173) were puchased from American Type Culture Collection (ATCC, Manassas, VA) and immediately cultured in Dulbecco's modified Eagle's medium (DMEM) #11965-084 (Life Technologies, Grand Island, NY) with 10% fetal bovine serum (FBS) #10437-028 (Life Technologies) and 1% penicillin/streptomycin/glutamine (P/S/G) #10378-016 (Life Technologies) mixture upon arrival. To differentiate the preadipocytes, 3T3-L1 cells were grown to 60% confluence and changed to DMEM (with 1% P/S/G) media with 10% calf serum #16170-078 (Life Technologies) for 48 hours. Differentiation was initiated by incubation with induction media containing DMEM with FBS, P/S/G, bovine insulin (I-5500; 1 μg/mL, Sigma, St. Louis, MO), dexamethasone (Sigma D-4902; 1 μM) and isobutylmethylxanthine (IBMX; Sigma I-5500; 115 μg/mL) for 48 hours. The cells were then further incubated with insulin media containing DMEM with FBS, P/S/G and insulin (1 microg/mL) for another 48-72 hours. When the cell confluence reached 90%, 3T3-L1 cells were serum-starved overnight followed by treatment with cathelicidin and/or other reagents to study the role of cathelicidin in lipid accumulation.
I5500
Manufactured by Merck Group
Sourced in United States
The I5500 is a laboratory equipment product offered by Merck Group. It is a high-performance instrument designed for a core function, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
I7018, by Merck Group (4 mentions)
Dexamethasone (dex), by Merck Group (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
6 well cell culture plate, by Corning (3 mentions)
3 isobutyl 1 methylxanthine 1 7018, by Merck Group (3 mentions)
Penicillin streptomycin glutamine (psg), by Thermo Fisher Scientific (2 mentions)
1 preadipocytes, by American Type Culture Collection (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
I5879, by Merck Group (2 mentions)
6 well tissue culture plate, by Corning (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Rosiglitazone, by Merck Group (2 mentions)
D1756, by Merck Group (2 mentions)
Oil red o, by Merck Group (2 mentions)
3 isobutyl 1 methyl xanthine (ibmx), by Merck Group (2 mentions)
Insulin, by Merck Group (2 mentions)
Dexamethasone d4902, by Merck Group (2 mentions)
William s e medium, by Thermo Fisher Scientific (1 mentions)
Percoll, by GE Healthcare (1 mentions)
32 protocols using i5500
1
Differentiation of 3T3-L1 Preadipocytes
2
Differentiation of 3T3-L1 Preadipocytes
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Mouse 3T3-L1 preadipocytes (#CL-173) were puchased from American Type Culture Collection (ATCC, Manassas, VA) and immediately cultured in Dulbecco's modified Eagle's medium (DMEM) #11965-084 (Life Technologies, Grand Island, NY) with 10% fetal bovine serum (FBS) #10437-028 (Life Technologies) and 1% penicillin/streptomycin/glutamine (P/S/G) #10378-016 (Life Technologies) mixture upon arrival. To differentiate the preadipocytes, 3T3-L1 cells were grown to 60% confluence and changed to DMEM (with 1% P/S/G) media with 10% calf serum #16170-078 (Life Technologies) for 48 hours. Differentiation was initiated by incubation with induction media containing DMEM with FBS, P/S/G, bovine insulin (I-5500; 1 μg/mL, Sigma, St. Louis, MO), dexamethasone (Sigma D-4902; 1 μM) and isobutylmethylxanthine (IBMX; Sigma I-5500; 115 μg/mL) for 48 hours. The cells were then further incubated with insulin media containing DMEM with FBS, P/S/G and insulin (1 microg/mL) for another 48-72 hours. When the cell confluence reached 90%, 3T3-L1 cells were serum-starved overnight followed by treatment with cathelicidin and/or other reagents to study the role of cathelicidin in lipid accumulation.
3
Isolation and Treatment of Primary Murine Hepatocytes
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Primary hepatocytes were isolated from mice using the collagenase type X (Wako, Japan; 039-17864) perfusion method.20 (link) Briefly, primary hepatocytes were isolated by collagenase type X (Wako Pure Chemical Industries, ltd, Japan; 039-17864) perfusion method. Cells were washed with hepatocyte wash medium (Thermo Fisher Scientific, Waltham, MA; 17704024), purified by Percoll (GE Healthcare, Chicago, IL; 17089101) density gradient separation, and resuspended in William’s E medium (Thermo Fisher Scientific; 12551032) with 5% fetal bovine serum, 10-nM dexamethasone, and 20-nM insulin. Cells were then seeded on collagen-coated plates at a final density of 3.5 × 104 cells/cm2. After 4 hours, attached cells were cultured with fresh medium and transduced with the indicated adenoviruses. Primary hepatocytes were treated for 4 hours with EBSS (Sigma-Aldrich, St. Louis, MO; E3024), containing 200-μM OA (16 hours; Sigma-Aldrich; O1008), 10-ng/mL TNF (16 hours; PeproTech), 1-μg/mL LPS (6 hours; Sigma-Aldrich; L2630), 50-μM SNAP (4 hours; Sigma-Aldrich, N3398), 100-nM PTIO (30 minutes; Sigma-Aldrich; P5084), and 20-μM CQ (Sigma-Aldrich, C6628). Insulin signaling in primary hepatocytes was stimulated with a 10-minutes exposure to 5-nM insulin (Sigma-Aldrich; I5500).
4
Cell Line Cultivation Protocols for HPV Research
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Ca Ski (HPV 16+), S12 (HPV 16+), SiHa (HPV 16+), HeLa (HPV 18+), C-33 A (HPV-negative), HEK293T (HPV-negative) and HaCaT (HPV-negative) cell lines were purchased from the American Type Culture Collection (ATCC). Ca Ski was maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS; Gibco, 10270–106) and 100 U/ml penicillin and streptomycin (Invitrogen, 15140163). SiHa, HeLa, HEK293T and C-33 A were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 100 U/ml penicillin and streptomycin (Invitrogen, 15140163). S12 and HaCaT cells were cultured in a 1:3 mixture of DMEM and Ham F-12 supplemented with 5% FBS (Gibco, 10270–106), 5 μg/ml insulin (Sigma-Aldrich, I5500), 8.4 ng/ml cholera toxin (Sigma-Aldrich, C8052), 24.3 μg/ml adenine (Solarbio, A8330), 10 ng/ml recombinant human epidermal growth factor (EGF; Solarbio, P0033) and 0.5 μg/ml hydrocortisone (Solarbio, G8450) (5 (link)). All cells were cultured in a humidified 37 °C incubator at 5% CO2 and passaged 2–3 times weekly as recommended by the ATCC.
5
Metabolic Profiling in Mice
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For the glucose tolerance test, mice that fasted overnight for 16 h were injected intraperitoneally (i.p.) with D-glucose (1 g*kg−1 body weight) (G8270, Sigma). For the insulin tolerance test, mice were injected i.p. with 1 U*kg−1 body weight of insulin (I-5500, Sigma) after 6 h of fasting. Tail blood was drawn at 15, 30, 60, 90, and 120 min postinjection. Blood glucose was measured using a glucometer (Accu-Check Guide, Roche) at each time point. Serum TG, AST, and ALT levels were measured using a Fuji Dry-Chem NX500i (Fujifilm, Japan). Serum insulin (EZRMI-13k, Millipore), glucose (BM-GLO-100, Biomax), and tissue glycogen (ab65620, Abcam) levels were measured using assay kits. Tail blood lactate was measured with a Lactate Pro 2 (LT-1730, Arkray) at each exercise start and end point. All experiments were performed according to the manufacturer’s instructions.
To measure the energy content in stool, bomb calorimetry was performed using 1 g of dried feces on an isoperibol calorimeter (6400EF, Parr Instrument).
To measure the energy content in stool, bomb calorimetry was performed using 1 g of dried feces on an isoperibol calorimeter (6400EF, Parr Instrument).
6
Breast Cancer Cell Differentiation Protocol
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Fresh scrapings were collected from primary breast cancer after surgical procedures in compliance with approved ethical permit. Permits were obtained from the regional ethics board at Karolinska Institutet in Stockholm and from the Stockholm medical biobank (ethical approval #02–061 with amendments 2013–1065–32 and 2015–2259; and ethical approval 2016/957–31 with amendment 2017/742–32). All patient material was anonymized. Single cells were generated immediately as previously described48 (link). Cells were then differentiated in DMEM/F-12 GlutaMAX medium (10565, Life Technologies) supplemented with 5% FBS, 10 ng/mL epidermal growth factor (EGF, PHG0311, Life Technologies), 5 μg/mL insulin (I5500, Sigma), 1 μg/mL hydrocortisone (H0888, Sigma), and 10 μg/mL cholera toxin (C8052, Sigma). All the experiments were performed within five generations of these cultures that were then discarded.
8
Isolation and Culture of Bovine Mammary Epithelial Cells
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MECs were isolated from the mammary tissue of Holstein cows.44 (link) Tissue blocks were washed several times using D-Hank's balanced salt solution containing dual antibodies as well as 75% alcohol. The tissue blocks were cut into small pieces using scissors, inoculated in cell culture flasks at an appropriate density and placed in a 37°C incubator with 5% CO2. The culture medium was changed every 48 h. The composition of the culture medium was DMEM/F12(C3132,VivaCell) containing 20% fetal bovine serum, 1% double antibiotic, bovine insulin (5 μg/mL,I5500,Sigma-Aldrich), and hydrocortisone (1 μg/mL,3867,Sigma-Aldrich) MECs were digested in stages using 0.25% trypsin to purify MECs according to the different sensitivities of MECs and fibroblasts to trypsin. Characterization of BMECs was achieved using CK18.
9
Amyloid Fibril Formation Assay
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A stock solution of 2mg ml−1 Congo Red (Sigma‐Aldrich 75768) was prepared in phosphate buffer and filtered three times using a 0.22 μm syringe filter (Millipore). A 2mg ml−1 bovine insulin (Sigma‐Aldrich I5500) was prepared in MilliQ water adjusted to pH 1.6 using concentrated HCl. The insulin sample was incubated overnight at 60°C. A 60 µl of each protein sample was added to a cuvette containing 1ml of buffer and 10 µl of the Congo Red stock solution. The samples were then allowed to incubate at room temperature for 30 min. A control spectrum containing only Congo Red was measured where 10 µl of the Congo Red stock solution was added to 1ml of buffer plus an additional 60 μl of buffer (to match the amount of protein added to each cuvette). Since the nTasA(±) samples contained multiple components, a UV‐vis spectrum (Cary 1E spectrophotometer) from 800 to 200nm was measured and the relative absorbance peaks at 280nm was used to ensure equal amounts of protein were measured between the two samples. The Congo Red spectra were acquired over a wavelength range of 400–700nm.
10
Adipogenic Differentiation of MSCs
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Adipogenic differentiation induction was performed as described previously (Watanabe et al., 2015 ). Briefly, when MSCs reached confluence, they were fed with complete adipogenic hormone cocktail: DMEM supplemented with 10% FBS, 10 μg/ml of insulin (I5500, Sigma), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX, I5879, Sigma) and 1 μM Dex (D4902, Sigma). The start point of differentiation was referred as day 0. Cells were then incubated in the same medium containing 5 μg/ml of insulin. After 2 days, the medium was replaced with a conditioned medium. Subsequently, the conditioned medium was changed regularly every 2 days. Intracellular lipid accumulation was evaluated using Oil Red O staining.
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