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Insulin Sigma I5500

Generation Of Neurospheres From Embryonic Stem Cells For Investigating The Temporal Specification Of Neural Stem/progenitor Cells

Generation Of Neurospheres From Embryonic Stem Cells For Investigating The Temporal Specification Of Neural Stem/progenitor Cells

Authors: Hayato Naka , Shiho Nakamura , Takuya Shimazaki & Hideyuki Okano Introduction This culture system, developed for the induction and selective amplification of neural stem/progenitor cells (NSPCs) from embryonic stem (ES) cells, allows the systematic characterization of their temporal specification. The use of ES cells in this system enables us to obtain a large number of NSPCs with early temporal identities and to control their regional identity by exposing them to extrinsic signals, recapitulating in vivo central nervous system (CNS) development. In this system, primary neurospheres derived from embryoid bodies (EBs) differentiate exclusively into neurons, and gliogenesis is activated in subsequent generations of neurospheres. The culture and differentiation assay take approximately 1 month to complete. Reagents EB3 ES cells (129/Ola-derived HPRT-negative E14tg2a ES cells carrying IRES-BSD in one Oct-3/4 locus, which allows for the selection of Oct-3/4-positive undifferentiated stem cells.) 293T cells Glasgow minimum essential medium (GMEM; Sigma G6148) MEM Non-essential amino acid solution (NEAA; Sigma M7145) Sodium pyruvate (Sigma S8636) Double processed tissue culture water (TC water; Sigma W3500) Hepes (Sigma H4034) Sodium bicarbonate (NaHCO3; Sigma S6297) 2-Mercaptoethanol (2ME; Sigma M7522) Blasticidin S hydrochloride (BlaS; Funakoshi KK-400) Trypsin-EDTA (0.25% trypsin, 1 mM EDTA) (Invitrogen/Gibco 25200-072) Dulbecco’s phosphate buffered saline 10 x (10 x PBS; Sigma D1408) Gelatin from porcine skin, Type A (Sigma G1890) Minimum essential medium alpha medium (DMEM; Invitrogen/Gibco 11900-016) Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen/Gibco 12100-038) F-12 Nutrient mixture (F12; Invitrogen/Gibco 21700-026) D-(+)-Glucose (Sigma G5767) L-Gl Continue reading >>

Figures And Data In Mitochondrial Coq Deficiency Is A Common Driver Of Mitochondrial Oxidants And Insulin Resistance | Elife

Figures And Data In Mitochondrial Coq Deficiency Is A Common Driver Of Mitochondrial Oxidants And Insulin Resistance | Elife

Proteomic analysis of adipocyte insulin resistance. (A) Adipose tissue 3H-2-DOG uptake during a glucose tolerance test in mice fed a high fat high sucrose diet (HFHSD) for indicated times. Results show meanS.E.M. of eight mice. *p<0.05 versus mice fed a HFHSD for 0 days, t-tests corrected for multiple comparisons. (B) 3T3-L1 adipocytes were treated with chronic insulin (CI), dexamethasone (Dex) or tumour necrosis factor- (TNF) to induce insulin resistance and stimulated with 100 nM insulin for 20 min where indicated before determination of insulin sensitivity by HA-GLUT4 abundance at the plasma membrane (PM). Results show meanS.E.M. of six separate experiments. *p<0.05 versus insulin-stimulated control cells, t-tests corrected for multiple comparisons. (C) Scatter plot of z-scores of protein changes (p<0.01) in adipose tissue from mice fed a HFHSD for 5 or 14 d (red=proteins consistently up-regulated, blue=proteins consistently down-regulated, purple=proteins with mixed response (up/down), orange=proteins altered at a single time point, black open circle=changed at both HFHSD time points and at least two in vitro models). (D) Three-dimensional direction analysis for proteomic data across the three 3T3-L1 adipocyte models of insulin resistance (coloured proteins= p < 0.01). Axes correspond to the z-scores of protein changes and coloured points indicate proteins changed in at least two out of three models (red=proteins up-regulated, blue=proteins down-regulated, purple=proteins with mixed response (up/down) across models). (E) Scatter plot of z-scores for pathways from proteomic analysis of insulin-resistant tissue (y-axis) or cells (x-axis). Selected pathways with z-scores>4 in tissue and cells are highlighted. (F) Heat map of correlations between expression of proteins Continue reading >>

Geo Accession Viewer

Geo Accession Viewer

Adipocyte differentiation was initiated 2 days after reaching confluence (Day 0, D0) by growing cells in DMEM supplemented with 10% Fetal Bovine Serum, 100U/mL penicillin and 100g/mL streptomycin, 1M dexamethasone (Sigma, Ref. D8893-1MG), 500M 3-isobutyl-1-methylxanthine (IBMX, Sigma, Ref. I5879) and 1g/mL bovine insulin (Sigma, Ref. I5500). Two days later (day 2, D2), medium was replaced by DMEM supplemented with 10% FBS, 1g/mL bovine insulin and a mix of 100U/mL penicillin and 100g/mL streptomycin (Life Technologies). Two days later (day 4, D4), medium was replaced by DMEM supplemented with 10% FBS and subsequently renewed every other day until day 9, D9. 3T3-L1 cells were transfected by siRNAs (100nM) using INTERFERin (Polyplus Transfection, Illkirch, France, Ref. 409-10). siRNAs were: control siRNA (Dharmacon, ON-TARGETplus non-targeting, ref. D-001810-10-10,) and Paral1-siRNA (Silencer Select ref. 101167, Ambion). Differentiated 3T3-L1 adipocytes were reverse-transfected as follows: cells were washed with 1x PBS, trypsinized and suspended (5x105 for 800L) in DMEM supplemented with 10% FBS containing 100U/mL penicillin and 100g/mL streptomycin. Cells in suspension were transfected by siRNA (100nM) with INTERFERin (Polyplus Transfection, Ref. 409-10) in 12-well plates according to the manufacturers instructions. 3T3-L1 cells were routinely grown at 37C and 5% CO2 in Dulbeccos Modified Eagles medium (DMEM)-high glucose (4.5g/L) containing 10% (v/v) Cosmic Calf Serum, 100U/mL penicillin and 100g/mL streptomycin. Seeding densities were as follows: 5x104 cells/well (12-well cluster), 12.5x104 (6-well cluster) or 5x105 cells (P100 dish). Proliferation medium was renewed every other days. Extract-All extraction of total RNA was performed according to the manufacturer's in Continue reading >>

Isolation And Growth Of Mouse Primary Myoblasts

Isolation And Growth Of Mouse Primary Myoblasts

Protocols See reference: Springer, M.L., T. Rando, and H.M. Blau (1997). "Gene delivery to muscle." In Current Protocols in Human Genetics. Unit 13.4, A. L. Boyle, ed. John Wiley & Sons, New York. Primary mouse myoblasts can be purified, stably transduced, and grown extensively in vitro. The transplantation of such cells can thus be used as a method of gene delivery. Primary cultures derived from skeletal muscle consist mainly of myoblasts and fibroblasts and the following protocol describes the purification of myogenic cells from such a mixed population. During the initial week or two of culturing, selective growth and passaging conditions allow the myoblasts to become the dominant cell type, and after two to three weeks in culture, nearly 100% of the cells stain positive for the myoblast specific protein desmin. Primary myoblasts can be isolated from mice of any age, but isolation from neonatal mice gives a greater yield of myogenic cells. Materials 70% ethanol in a squirt bottle Sterile phosphate buffered saline (PBS) Collagenase/dispase/CaCl2 solution F-10-based primary myoblast growth medium F-10/DMEM-based primary myoblast growth medium Fusion medium Neonatal mice, preferably 1-3 days old Sharp curved surgical scissors 2 pairs of fine forceps Low power stereo dissecting microscope Sterile razor blade 80 m nylon mesh (e.g. Nitex; Tetko, Inc.; Monterey Park, CA) Small sterile funnel Collagen-coated tissue culture dishes: 35 mm, 60 mm, 100 mm, and 150 mm sizes Humidified 37°C, 5% CO2 incubator Inverted microscope Isolate limb muscle Sacrifice 1-5 neonatal mice by decapitation or CO2 inhalation. Rinse the limbs with 70% ethanol and remove them with sterile scissors. Dissect the muscle away from the skin and bone with sterile forceps. Dissection is easier if done unde Continue reading >>

Sigma-i5500 Insulin 27 Usp Units/mg 100mg--/--

Sigma-i5500 Insulin 27 Usp Units/mg 100mg--/--

-Sigma-AldrichSigmaSupelcoRdHFlukaAldrich. Sigma-E5389 Erythromycin Cell Culture 1G Sigma-E5389 Erythromycin Cell Culture 5G Sigma-527254 Trimethylsilyldiazomethane solution 25ml Sigma-527254 Trimethylsilyldiazomethane solution 100ml Sigma-01696 Acrylamide 99.5% 100G Sigma-01696 Acrylamide 99.5% 250G Sigma-01696 Acrylamide 99.5% 500G Sigma-01897 5,- ADP Na2 Adenosine 5,-diphosphate disodium 1g Sigma-01930 5,- AMP Na2 Adenosine 5,-monophosphate disodium 1G Sigma-01930 5,- AMP Na2 Adenosine 5,-monophosphate disodium 5G Sigma-02055 5,- ATP Na2 Adenosine 5,-triphosphate disodium 1G Sigma-02055 5,- ATP Na2 Adenosine 5,-triphosphate disodium 5G Sigma-03609 Ethylenediaminetetraacetic Acid EDTA 100G Sigma-03780 EGTA Ethylene Glycol-bis2-aminoethylether-N,N,N,N-Tetraacetic Acid 5G Sigma-03780 EGTA Ethylene Glycol-bis2-aminoethylether-N,N,N,N-Tetraacetic Acid 25G Sigma-03780 EGTA Ethylene Glycol-bis2-aminoethylether-N,N,N,N-Tetraacetic Acid 25G Sigma-05500 8GX :8GX Alcian Blue 8GX 1G Sigma-05500 8GX :8GX Alcian Blue 8GX 5g Sigma-05500 8GX :8GX Alcian Blue 8GX 25G Sigma-06232 6 Aluminum Chloride Hexahydrate 100G Sigma-06232 6 Aluminum Chloride Hexahydrate 500G Sigma-06420 CG120 Amberlite CG120 50g Sigma-06428 IR120 Amberlite IR120 Hydrogen Form 250G Sigma-06429 IRC50 Amberlite IRC50 Hydrogen Form 250G Sigma-06433 IRA410 Amberlite IRA410 chloride form 250G Sigma-06435 CG50 Amberlite CG50 50G Sigma-06438 CG400 Amberlite CG400 50G Sigma-06469 CG-120-II Amberlite CG-120-II 50G Sigma-06471 CG-400-II Amberlite CG-400-II 50G Sigma-06563 Methotrexate 99% 10MG Sigma-07549 -1- PyBOP 25G Sigma-07549 -1- PyBOP 100g Sigma-07549 -1- PyBOP 500G Sigma-09658 TCFH Chloro-N,N,N,N,-Tetramethylformamidinium Hexafluorophosphate 1g Sigma-09658 TCFH Chloro-N,N,N,N,-Tetramethylformamidinium Hexafluoropho Continue reading >>

The Signalling Conformation Of The Insulin Receptor Ectodomain

The Signalling Conformation Of The Insulin Receptor Ectodomain

The signalling conformation of the insulin receptor ectodomain Nature Communicationsvolume9, Articlenumber:4420 (2018) | Download Citation Understanding the structural biology of the insulin receptor and how it signals is of key importance in the development of insulin analogs to treat diabetes. We report here a cryo-electron microscopy structure of a single insulin bound to a physiologically relevant, high-affinity version of the receptor ectodomain, the latter generated through attachment of C-terminal leucine zipper elements to overcome the conformational flexibility associated with ectodomain truncation. The resolution of the cryo-electron microscopy maps is 3.2 in the insulin-binding region and 4.2 in the membrane-proximal region. The structure reveals how the membrane proximal domains of the receptor come together to effect signalling and how insulins negative cooperativity of binding likely arises. Our structure further provides insight into the high affinity of certain super-mitogenic insulins. Together, these findings provide a new platform for insulin analog investigation and design. The human insulin receptor is a homodimeric, disulphide-linked ()2 receptor tyrosine kinase. Despite the significance of the receptors signaling in a number of major disease states, a complete, atomic-level understanding of the way in which insulin binds to the receptor and effects signal transduction has proved elusive 1 . An obstacle is that the isolated, soluble receptor ectodomain (sIR), the entity most amenable to structural biology investigation, lacks the high affinity and the negative cooperativity of insulin binding that is characteristic of the holo-receptor (hIR) 2 , 3 . In particular, sIR binds two insulin molecules with equal nanomolar affinity with no negative coope Continue reading >>

Insulin Injection And Hemolymph Extraction To Measure Insulin Sensitivity In Adult Drosophila Melanogaster

Insulin Injection And Hemolymph Extraction To Measure Insulin Sensitivity In Adult Drosophila Melanogaster

Protocol 1. Insulin Solution Preparation Prepare fresh bovine insulin solution by dissolving insulin in PBS to achieve the concentration of 0.01 mg/ml. Both insulin/PBS and control PBS solutions must be kept on ice throughout the injection procedure. These solutions should be prepared with 0.5% (v/v) FD&C Blue no. 1 food coloring. 2. Needle Preparation and Injection Set-up Prepare capillary glass needles using a micropipette puller.The following puller settings produce needles of sufficient quality for injection: Heat, 345; Pull, 210; Velocity, 100; Time, 200 (100ms). Freshly pulled needles must be blunted by pressing the tip through a Kimwipe tissue. This blunting process removes the elongated high resistance tip and produces a stouter tip with a larger pore diameter. Place blunted needles tip-up in a microcentrifuge tube containing the insulin/PBS solution or PBS alone. Capillary action results in back-filling of each needle. Observe the tip of the needle under a stereomicroscope to ensure that no debris or air bubbles appear at the tip. Discard any needles that do not fill cleanly. Insert filled needles into the manual microinjector needle holder and position the needle holder with a micromanipulator so that the needle tip is visible through a stereomicroscope. Apply positive pressure on the fluid column in the needle by turning the manual microinjector micrometer knob attached to the plunger on the microinjector syringe. Verify that sufficient pressure for fluid displacement has been applied by touching a Kimwipe to the tip of the needle and confirming fluid flow. Once the needle has been prepared for injection, affix a calibration chart to the needle shaft with clear tape, and bring the needle tip into focus through a stereomicroscope under 20X magnification. 3. Fl Continue reading >>

Chronic Hyperinsulinemia Reduces Insulin Sensitivity And Metabolic Functions Of Brown Adipocyte

Chronic Hyperinsulinemia Reduces Insulin Sensitivity And Metabolic Functions Of Brown Adipocyte

Abstract The growing pandemics of diabetes have become a real threat to world economy. Hyperinsulinemia and insulin resistance are closely associated with the pathophysiology of type 2 diabetes. In pretext of brown adipocytes being considered as the therapeutic strategy for the treatment of obesity and insulin resistance, we have tried to understand the effect of hyperinsulinemia on brown adipocyte function. We here with for the first time report that hyperinsulinemia-induced insulin resistance in brown adipocyte is also accompanied with reduced insulin sensitivity and brown adipocyte characteristics. CI treatment decreased expression of brown adipocyte-specific markers (such as PRDM16, PGC1α, and UCP1) and mitochondrial content as well as activity. CI-treated brown adipocytes showed drastic decrease in oxygen consumption rate (OCR) and spare respiratory capacity. Morphological study indicates increased accumulation of lipid droplets in CI-treated brown adipocytes. We have further validated these findings in vivo in C57BL/6 mice implanted with mini-osmotic insulin pump for 8weeks. CI treatment in mice leads to increased body weight gain, fat mass and impaired glucose intolerance with reduced energy expenditure and insulin sensitivity. CI-treated mice showed decreased BAT characteristics and function. We also observed increased inflammation and ER stress markers in BAT of CI-treated animals. The above results conclude that hyperinsulinemia has deleterious effect on brown adipocyte function, making it susceptible to insulin resistance. Thus, the above findings have greater implication in designing approaches for the treatment of insulin resistance and diabetes via recruitment of brown adipocytes. Continue reading >>

Wo2016176935a1 - Leptin Active Peptide Having Cd Loop And E Helix Region Mutations, Coding Gene Thereof, And Application Thereof - Google Patents

Wo2016176935a1 - Leptin Active Peptide Having Cd Loop And E Helix Region Mutations, Coding Gene Thereof, And Application Thereof - Google Patents

WO2016176935A1 - Leptin active peptide having cd loop and e helix region mutations, coding gene thereof, and application thereof - Google Patents Leptin active peptide having cd loop and e helix region mutations, coding gene thereof, and application thereof WO2016176935A1 PCT/CN2015/088554 CN2015088554W WO2016176935A1 WO 2016176935 A1 WO2016176935 A1 WO 2016176935A1 CN 2015088554 W CN2015088554 W CN 2015088554W WO 2016176935 A1 WO2016176935 A1 WO 2016176935A1 Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.) A61MEDICAL OR VETERINARY SCIENCE; HYGIENE A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES A61K38/00Medicinal preparations containing peptides A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans A61K38/2264Obesity-gene products, e.g. leptin C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans C07K14/5759Products of obesity genes, e.g. leptin, obese (OB), tub, fat A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING A01KANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS A01K2217/05Animals comprising random inserted nucleic acids (transgenic) A61MEDICAL OR VETERINARY SCIENCE; HYGIENE A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PU

Sigma Insulin I5500-1

Sigma Insulin I5500-1

Sigma insulin I5500-1 SIGMA-ALDRICH zuiRNAisigmasigma sigma 51370 Harmane 1G sigma 222984 dl-Propranolol HCl 5G sigma 284785 S-6-Methoxy-a-Methyl-2-Naphthaleneacetic acid 25G sigma 857416 3-3,4-Dihydroxyphenyl-2-methyl-L-Alanine 5g sigma 857440 Chloramphenicol 25G sigma A4669 Acycloguanosine 500mg sigma A5882 Antipyrine 500G sigma A7655 Atenolol 25G sigma A8523 Amoxicillin 25g sigma B7283 Benserazide HCl 5G sigma C2505 Corticosterone 500MG sigma C4024 Carbamazepine 25G sigma C4216 Coumarin 500G sigma C4255 Creatinine 1KG sigma C4522 Cimetidine 25G sigma C5793 Ceftriaxone Na 1G sigma C6022 Cyproheptadin HCl 1G sigma D1756 Dexamethasone 500MG sigma D2521 Diltiazem 10G sigma D3900 Desipramine 25G sigma D4007 Phenytoin 500G sigma D6270 1-2,6-dichlorobenzyl-ideneaminoguanidine 100MG sigma D9628 l-3,4-Dihydroxyphenyl-alanine 100G sigma E6888 Enalapril Maleat 5G sigma F4381 Furosemide 25G sigma F8514 Flurbiprofen 25G sigma F9677 Felodipine 25MG sigma H0888 Hydrocortisone 10G sigma H4759 Hydrochlorothiazide 100G sigma I0899 Imipramine HCl 25G sigma K1751 Ketoprofen 100G sigma M5391 Metoprolol x 1/2 Tartrate 10G sigma N144 Nitrendipine 500MG sigma N7758 Naloxone 1G sigma N8280 Naproxen 5G sigma N9890 Norfloxacin 25G sigma P0847 Piroxicam 10G sigma P7412 Pirenzepine 1G sigma Q3625 Quinidine 25G sigma R101 Ranitidine 5G sigma S0883 Sulfasalazine 100G sigma S8010 Sulpiride 100G sigma T1500 Testosterone 25G sigma T1633 Theophylline 1KG sigma T2528 Terbutaline x 1/2 Tartrate 5G sigma T69809 Trimethoxybenzamide HCl 100G sigma V4629 Verapamil 10G sigma E3763 Ethoxyresorufin 5MG sigma A2500 Phenacetin 100G sigma UC175 S-+Mephenytoin 5MG sigma T1912 Paclitaxel 25MG sigma UC214 S-Warfarin 10MG sigma D6899 Diclofenac sodium salt 10G sigma O104 Omeprazole 500MG sigma D9684 Dextromethorphan Continue reading >>

Disulfide Bridges Remain Intact While Native Insulin Converts Into Amyloid Fibrils

Disulfide Bridges Remain Intact While Native Insulin Converts Into Amyloid Fibrils

Go to: Protein aggregates play an important role in living cells due to their ubiquity. Aggregation of proteins results in the formation of long, unbranched β-sheet-rich structures, commonly known as amyloid fibrils [1]. These fibrils are found as deposits in the tissues and organs of patients with various amyloid-associated diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), prion disease, and type II diabetes [2], [3]. There is also increasing evidence that small aggregates of misfolded proteins are most toxic and the formation of amyloid fibrils is a defense mechanism [4]. It is known that more than 20 proteins that can aggregate to form amyloid-like fibrils. Previously, it was proposed that the ability to form amyloid fibrils is not a peculiarity of this small group of disease-related proteins, but rather, the ability to form amyloids is a generic property of the polypeptide chain [5]. Thus, many physiochemical properties of protein sequences, such as charge, hydrophobicity, and the tendency to form secondary structures, were extensively elucidated in recent decades to understand their relative propensities for amyloid fibril formation. One example of these properties is disulfide bonds, which are present in 65% of all secreted proteins, and in 50% of proteins involved in amyloidosis [6]. The behavior of disulfide bonds upon protein aggregation has been extensively studied over the past decade [7], [8], [9]. Disulfide bonds limit the way in which a protein or a peptide can aggregate into a fibril via steric restraint. For example, the reduction of intra-molecular disulfide bonds in β2 microglobulin was determined to limit the formation of long fibrils upon protein aggregation [9], [10]. In our previous work, we de Continue reading >>

Geo Accession Viewer

Geo Accession Viewer

Bovine insulin (I5500, Sigma-Aldrich, Stockholm, Sweden) was added to the medium during the IVM period of 22 hours, at either 0.1 g/mL (INS0.1) or 10 g/mL (INS10). The two different insulin concentrations INS10 and INS0.1 were used in this study in purpose to investigate a potential dose-dependent effect of insulin during oocyte maturation. Briefly, abattoir-derived cumulus oocyte complexes (COCs) (n=882) were matured in groups of 30-40 in vitro for 22 h in 500 l serum-free maturation medium (IVM). IVM medium, consisted of bicarbonate-buffered TCM199 (M2154) supplemented with 0.68 mM L-glutamine (G8540), 0.5 mg/mL FSH and 0.1 mg/mL LH (Stimufol; PARTNAR Animal Health, Port Huron, Canada), 50 mg/mL gentamicin and 0.4% w/v BSA. Bovine insulin (I5500) was added to the medium at either 0.1 g/mL (INS0.1) or 10 g/mL (INS10). A control group without insulin supplementation (INS0) was run in parallel during each production week, medium. Matured oocytes were fertilized in vitro with sperm from a dairy bull with proven field and in vitro fertility and presumed zygotes were incubated under humidified atmosphere of 5 % CO, 5% O and 90 % N at 38.5C in 500 l modified synthetic oviductual fluid per well until Day 8. Resulting blastocysts (BC8) were evaluated (developmental stage and quality grade) and washed three times in PBS-PVA before individually frozen at -80C. gDNA was extracted from pools of 10 blastocysts using the AllPrep DNA/RNA micro kit (Qiagen). The methylation analysis was performed using EmbryoGENE DNA methylation array (EDMA) platform. The methodology have been thoroughly describef in (Saadi et al., 2014). Briefly, 10ng of DNA sample and spike-in control were cut in 160bp fragment using MSEI restriction enzyme. MseLig 12 and MseLig 21 adapters were added to the fragme Continue reading >>

Ablation Of Ng2 Proteoglycan Leads To Deficits In Brown Fat Function And To Adult Onset Obesity

Ablation Of Ng2 Proteoglycan Leads To Deficits In Brown Fat Function And To Adult Onset Obesity

Abstract Obesity is a major health problem worldwide. We are studying the causes and effects of obesity in C57Bl/6 mice following genetic ablation of NG2, a chondroitin sulfate proteoglycan widely expressed in progenitor cells and also in adipocytes. Although global NG2 ablation delays early postnatal adipogenesis in mouse skin, adult NG2 null mice are paradoxically heavier than wild-type mice, exhibiting larger white fat deposits. This adult onset obesity is not due to NG2-dependent effects on CNS function, since specific ablation of NG2 in oligodendrocyte progenitors yields the opposite phenotype; i.e. abnormally lean mice. Metabolic analysis reveals that, while activity and food intake are unchanged in global NG2 null mice, O2 consumption and CO2 production are decreased, suggesting a decrease in energy expenditure. Since brown fat plays important roles in regulating energy expenditure, we have investigated brown fat function via cold challenge and high fat diet feeding, both of which induce the adaptive thermogenesis that normally occurs in brown fat. In both tests, body temperatures in NG2 null mice are reduced compared to wild-type mice, indicating a deficit in brown fat function in the absence of NG2. In addition, adipogenesis in NG2 null brown pre-adipocytes is dramatically impaired compared to wild-type counterparts. Moreover, mRNA levels for PR domain containing 16 (PRDM16) and peroxisome proliferator-activated receptor γ coactivator (PGC)1-α, proteins important for brown adipocyte differentiation, are decreased in NG2 null brown fat deposits in vivo and NG2 null brown pre-adipocytes in vitro. Altogether, these results indicate that brown fat dysfunction in NG2 null mice results from deficits in the recruitment and/or development of brown pre-adipocytes. As Continue reading >>

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Serial passaging and transduction with recombinant lentiviruses carrying hTERT gene The immortalized human mammary progenitor cells hTERT (K5+/K19+) is a clonal cell population of progenitors coexpressing normal mammary and stem cell markers. It has the ability to self-renew and differentiate into luminal and/or myoepithelial cell lineages. Through propagation in different optimized media, the cells are able to give rise to new population of cells with different morphology and characteristics, such as mucin-1 positive cells, vimentin-negative cells, or expressing basal, luminal, and other stem cell markers. Aldehyde dehydrogenase 1A3 enzyme is noticeably higher in expression, a marker commonly used for isolating normal and tumor mammary stem cells. K5+/K19+cells have Wnt, Notch, and hedeghog gene regulatory pathways. It is a valuable tool in research in the field of biology, the stem/progenitor origin, and the heterogeneity mechanisms in breast cancer. Use of PriCoat T25 Flasks ( G299 ) or Applied Cell Extracellular Matrix ( G422 ) is required for cell adhesion to the culture vessels. Grow cells in ECM-coated culture vessels with the following conditions.To make DFCI-1 the base medium is Prigrow VIII and Prigrow IV (1:1, vol/vol) medium available from ABM ( TM018 and TM004 ). To make the completed growth medium, add the following components to the base medium at final concentration: 0.01M HEPES (Gibco; 15630), 1% fetal bovine serum ( TM999 ), 12.5 ng/ml (EGF) epidermal growth factor ( Z100135 ), 6.5 ng/ml triiodothyronine (Sigma; T5516), 0.545 ng/ml beta-estradiol (Sigma; E2257), 1 g/ml insulin (Z101065), 1 g/ml hydrocortisone (Sigma; I5500), 0.006X ethanolamine (Sigma; E9508), 14.1 g/ml phosphoethanolamine (Sigma; P0503), 10 g/ml transferrin (Sigma; T2252), 2 mM gluta Continue reading >>

Procedures

Procedures

Procedures for Culture of Human Mammary Epithelial Cells Feeding and Plating Volumes and Densities C. Subculture of Primary Organoid Cultures (references: Garbe et al. 2009, LaBarge et al. 2103, Hammond et al. 1984, Stampfer 1985) Basal M87A can be purchased from the UCSF Cell Culture Facility. Contact Amy Choi, media production supervisor, at [emailprotected] ; 415-476-8686. You will need to add supplements before use. If you want to make up your own M87A please contact us for more information. MCDB 170-like media can be purchased from Lonza (MEBM, MEGM) or ThermoFisher (HuMEC medium, Medium 171). Maintenance of the appropriate pH is critically important. Media color should be salmon-pink. If it gets too acid or basic it will diminish the cells growth potential. Fetal calf serum (we order from ThermoFisher Scientific; cat# 26140) 100 ml bottles of FBS can be thawed and kept at 4C for ongoing use. Larger volumes can be aliquoted and refrozen for storage at -20C/-80C. BPE (Bovine Pituitary Extract): we order Hammond Cell Tech cat#1078-NZ Stock BPE is at ~14 mg/ml. Final concentration in medium is ~35 g/ml. Take bottles (100 or 200 mL) of "raw" BPE out of freezer and put in 37 C water bath just until thawed. Transfer BPE to several Oak Ridge tubes and balance pairs against each other. KEEP BPE ON ICE AT ALL TIMES DURING THIS PROCEDURE. Centrifuge at 15K rpm for 30 min. at 4C. Pour the supernatant into clean Oak Ridge tubes and centrifuge again at 15K for 20 min. If supernatant doesn't appear to be clear, centrifuge 10 min. more, same conditions. Transfer supernatant from all tubes into flask. IT SHOULD BE FREE OF ANY OBVIOUS DEBRIS AND APPEAR CLEAR, otherwise filters will immediately clog up. Discard the pellets at the bottom of the tubes. Filter the supernatant through Continue reading >>

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