3T3-L1 cells (American Type Culture Collection®, CL-173) were cultured in Dulbecco’s Modification of Eagle’s Medium (DMEM) with 1 g/L glucose (Corning®, 10–014-CV), supplemented with 10% foetal bovine serum (FBS) (Thermo Fisher Scientific, 12,483,020), 1% Penicillin-Streptomycin (GibcoTM, 15,140,122), and 1% L-glutamine (GibcoTM, 25,030,081), at 37°C in a humidified CO2 incubator. For differentiation, 3T3-L1 cells were seeded at 30,000 cells/cm2 in the media described above, placed in the incubator for 48 hours, to reach 100% confluence, and differentiated with DMEM 1X with 4.5 g/L glucose (Corning®, 10–013-CV), supplemented with 10% FBS, 1% Penicillin-Streptomycin, 1% L-glutamine, 1 μg/ml insulin (insulin solution from bovine pancreas in 25 mM HEPES, Sigma-Aldrich, I0516-5ML), 0.25 μM dexamethasone (DEX) (Sigma-Aldrich, D4902-100 MG) and 0.5 mM isobutyl-methyl-xanthine (IBMX) (Sigma-Aldrich, I5879-250 MG) (Figure 1 ). 48 hours following initiation of differentiation, the media was replaced with maintenance media (DMEM with 4.5 g/L glucose, 10% FBS, 1% penicillin streptomycin, 1% L-glutamine, and 1 µg/ml insulin), and the cells were re-supplemented with maintenance media every 48 hours until day 21 (Figure 1 ).
I0516 5ml
Manufactured by Merck Group
I0516-5ML is a laboratory reagent produced by Merck Group. It is a 5 milliliter solution intended for use in various scientific applications. The core function of this product is to serve as a reference material or chemical standard for analytical procedures. No additional details about the intended use or composition of this product can be provided in an unbiased and factual manner.
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Lab products found in correlation
D4902 100mg, by Merck Group (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Dmem hg, by Thermo Fisher Scientific (1 mentions)
Dmem 1x, by Thermo Fisher Scientific (1 mentions)
Crl 1772, by American Type Culture Collection (1 mentions)
Dmem hg, by Cytiva (1 mentions)
Fetal bovine serum (fbs), by Bovogen (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Htb 112, by American Type Culture Collection (1 mentions)
Colchicine, by Merck Group (1 mentions)
L glutamine and hepes, by Thermo Fisher Scientific (1 mentions)
Cytochalasin d, by Merck Group (1 mentions)
S8398, by Merck Group (1 mentions)
Mouse monoclonal anti α tubulin clone dm 1a, by Abcam (1 mentions)
Anti histone h3 ab1791, by Abcam (1 mentions)
6 protocols using i0516 5ml
1
3T3-L1 Cell Differentiation Protocol
2
Insulin Resistance Induction in C2C12 Myoblasts
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(murine myoblasts, CRL-1772, ATCC) were seeded at a density of 5 × 105 cells in 60 mm plates and maintained in Dulbecco’s modified Eagle’s high-glucose medium (DMEM-HG, SH30243.01, Hyclone Laboratories) supplemented with 10% fetal bovine serum (FBS, 1400–500, Seradigm) for 24 h. Thereafter, C2C12 cells were differentiated in DMEM-HG supplemented with 0.2% FBS for 48 h. To induced insulin resistance, differentiated cells were incubated with DMEM-1X (11966025, Thermo Fisher Scientific) supplemented with 5 mM glucose and 0.75 mM sodium palmitate for 18 h. Palmitate-containing media was prepared as previously described51 (link). Controls were cultured in the absence of palmitate. To examine insulin signaling, cells were treated with either vehicle or 100 nM insulin (I0516-5ML, Sigma-Aldrich) for 15 min. Thereafter, cells were washed with phosphate-buffered saline (PBS, 20-031-CV, Corning) and harvested in PBS followed by centrifugation at 10,000 × g for 10 min at 4 °C. Cell pellets were used for RNA isolation and protein analysis.
3
Glucose and Insulin Tolerance Tests in Mice
4
Endometrial Cell Line Treatments
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HEC1A cells, a non-receptive human endometrial adenocarcinoma cell line (ATCC HTB-112™), were grown at 37°C in 5% CO 2 in McCoy's 5A Medium (1×)/l-glutamine (GIBCO) supplemented with 10% fetal bovine serum (Bovogen Biologicals Pty, Essendon, VIC, Australia) and 1% streptomycin and penicillin (Invitrogen) until confluent.
RL95-2 cells, a receptive human endometrial adenocarcinoma cell line (ATCC CRL-1671™), were grown at 37°C in 5% CO 2 in DMEM/F-12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12) containing HEPES and L-glutamine (GIBCO) supplemented with 10% fetal bovine serum (Bovogen Biologicals Pty), 0.005 mg/mL insulin (I0516-5ML, Sigma Aldrich) and 1% streptomycin and penicillin (Invitrogen) until confluent.
Confluent cells were treated with either colchicine (3 µg/ mL; Sigma Aldrich) or cytochalasin D (3 µg/mL; Sigma Aldrich) for 18 h and washed with PBS to deactivate the treatment.
RL95-2 cells, a receptive human endometrial adenocarcinoma cell line (ATCC CRL-1671™), were grown at 37°C in 5% CO 2 in DMEM/F-12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12) containing HEPES and L-glutamine (GIBCO) supplemented with 10% fetal bovine serum (Bovogen Biologicals Pty), 0.005 mg/mL insulin (I0516-5ML, Sigma Aldrich) and 1% streptomycin and penicillin (Invitrogen) until confluent.
Confluent cells were treated with either colchicine (3 µg/ mL; Sigma Aldrich) or cytochalasin D (3 µg/mL; Sigma Aldrich) for 18 h and washed with PBS to deactivate the treatment.
5
Insulin-Induced Glucose Monitoring in Mice
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Fed mice were injected with 1 unit/kg body weight insulin (Sigma, I0516-5ML) intraperitoneally. Tail vein blood was used to monitor blood glucose levels at different time points: 0 (before glucose injection), 15, 30, 60, 90, and 120 min after glucose injection using a blood glucose meter (Contour, Bayer).
6
Drosophila Cell Line Maintenance and Antibody Usage
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The ML-D17c3 cell line, stock 107, was obtained from the Drosophila Genomics Resource Center cell line repository (http://dgrc.cgb.indiana.edu/ ). Cells were grown on pretreated tissue culture dishes with extracellular matrix as previously described (Currie and Rogers 2011 (link)) in M3 media (S-8398, Sigma-Aldrich) containing 1 mg/mL of yeast extracts (Y-1000, Sigma-Aldrich) and 2.5 mg/mL of bactopeptone (211677, Difco), 10% FBS (SH30070.02, Hyclone), and 10 mg/mL of insulin (I0516-5ml Sigma-Aldrich). The HepG2 cell line was kindly provided by B. Graveley (Institute for Systems Genomics, UCONN Health Center, Farmington) and maintained in Dulbecco's modified Eagle's medium (DMEM) (SH30022.01, Hyclone) supplemented with 10% FBS and 1% penicillin/streptavidin (15140-163, Invitrogen).
Rabbit polyclonal anti-Histone H3 (ab1791) and mouse monoclonal anti-α-tubulin (clone DM1A) were obtained from Abcam and Sigma-Aldrich, respectively. Mouse monoclonal anti-KDel (ADI-SPA-827), mouse monoclonal anti-Shot (mABRod1), and mouse monoclonal anti-ninein (clone F-5) were purchased from Enzo Life Sciences, the Developmental Studies Hybridoma Bank and Santa Cruz, respectively.
Rabbit polyclonal anti-Histone H3 (ab1791) and mouse monoclonal anti-α-tubulin (clone DM1A) were obtained from Abcam and Sigma-Aldrich, respectively. Mouse monoclonal anti-KDel (ADI-SPA-827), mouse monoclonal anti-Shot (mABRod1), and mouse monoclonal anti-ninein (clone F-5) were purchased from Enzo Life Sciences, the Developmental Studies Hybridoma Bank and Santa Cruz, respectively.
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