Insulin

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Insulin is a lab equipment product designed to measure and analyze insulin levels. It provides accurate and reliable results for research and diagnostic purposes.

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Insulin from bovine pancreas (52 citations) Insulin solution (35 citations) Bovine pancreas insulin (11 citations) Insulin-transferrin-selenium mix (4 citations) Human Insulin ELISA (3 citations) Insulin from porcine pancreas (3 citations) Insulin antibody (2 citations) Bovin Insulin (2 citations) Insulin-transferrin-sodium selenite medium supplement (2 citations) Insulin-transferrin sodium selenite solution (2 citations)

4 936 protocols using insulin

Adipocyte Differentiation Protocol

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For collected iWAT samples, the connective fiber and blood vessels were removed and washed three times with PBS buffer containing 200 U/mL penicillin (Sigma, St. Louis, USA) and 200 U/mL streptomycin (Sigma, St. Louis, USA). The adipocyte culture was carried out according to our previous publication. Briefly, pre-adipocytes were seeded onto 35-mm culture dishes at a density of 8 × 10⁴ cells/dish, and incubated at 37 °C under a humidified atmosphere of 5% CO₂ and 95% air until confluence. Differentiation of white pre-adipocytes was performed as following. Cells grown to 100% confluence (Day 0) were exposed to the induction DMEM/F12 containing dexamethasone (1 μM; Sigma), insulin (10 μg/mL; Sigma), IBMX (0.5 mM, Sigma) and 10% FBS. Four days after the induction (from Day 2), cells were maintained in the induction medium containing insulin (10 μg/mL, Sigma) and 10% FBS until the day for cell harvest. And brown adipocytes were induced to differentiate using DEME/F12 with 15% FBS, 0.5 mM IBMX; 0.25 mM indomethacin; 2 μg/mL dexamethasone; 1 nM T3, 20 nM insulin for 2 days and subsequently maintained on differentiation media (1 nM T3, 20 nM insulin).

Gan L., Liu Z., Chen Y., Dan L.u.o., Feng F., Liu G, & Sun C. (2016). α-MSH and Foxc2 promote fatty acid oxidation through C/EBPβ negative transcription in mice adipose tissue. Scientific Reports, 6, 36661.

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Culturing IBC Cell Lines with Supplements

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All IBC cell lines were grown in HAMS F12 media (Gibco 11765–054) in a 5% CO₂ incubator. Media for SUM-149 cells was supplemented with 5% FBS (Atlanta Biologicals), 10mM HEPES (Thermo Fisher 15630080), 1x antibiotic-antimycotic (anti-anti, Thermo Fisher 15240062), 1μg/mL hydrocortisone (Sigma H4001), and 5μg/mL insulin (Sigma I9278). SUM-190 media was supplemented with 1% FBS, 1μg/mL hydrocortisone, 5μg/mL insulin (Sigma I0516), 50nM sodium selenite (Sigma S9133), 5μg/mL apo-Transferrin (Sigma T-8158), 10nM triiodo thyronine (T3, Sigma T5516), 10mM HEPES, and 0.03% ethanolamine (Sigma 411000). MDA-IBC-3 cells were grown with 10% FBS, 1μg/mL hydrocortisone, 1x anti-anti, and 5μg/mL insulin (Sigma I0516). SUM cell lines were obtained from Stephen Ethier at the Medical University of South Carolina, and MDA-IBC-3 cells were obtained directly from Wendy Woodward at the University of Texas MD Anderson Cancer Center. All cell lines were routinely tested for mycoplasma contamination (Lonza LT07–418) and were authenticated using fragment analysis at the University of Michigan DNA sequencing core. Olaparib (MedChem Express HY-10162) and veliparib (MedChem Express HY-10129) were reconstituted in 100% DMSO for cellular assays.

Michmerhuizen A.R., Pesch A.M., Moubadder L., Chandler B.C., Wilder-Romans K., Cameron M., Olsen E., Thomas D.G., Zhang A., Hirsh N., Ritter C.L., Liu M., Nyati S., Pierce L.J., Jagsi R, & Speers C. (2019). PARP1 Inhibition Radiosensitizes Models of Inflammatory Breast Cancer to Ionizing Radiation. Molecular cancer therapeutics, 18(11), 2063-2073.

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Culturing Breast Cancer Cell Lines

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The human breast cancer cell lines MDA-MB-231 and MCF-7 were obtained from American Type Culture Collection (Manassas, VA, USA). MDA-MB-231 cells were cultured in RPMI1640 medium (Hyclone, LA, USA) with 10% fetal bovine serum (FBS, Gibco, NYC, USA) at 37 °C in 5% CO₂ incubator. MCF-7 cells were grown in MEM medium (Hyclone, LA, USA) with 10% FBS and 0.01 mg/ml insulin (Sigma-Aldrich, SL, USA) at 37 °C in 5% CO₂ incubator. Before insulin stimulation, cells were pre-seeded in plates with insulin-free medium for 24 h. insulin was purchased from Sigma-Aldrich. insulin was dissolved in HCI solution (pH 2.0). The final concentrations of HCI solution (pH 2.0) in the culture medium were less than 0.1%, and these amounts were also included in the corresponding controls.

Xia B., Hou L., Kang H., Chang W., Liu Y., Zhang Y, & Ding Y. (2020). NR2F2 plays a major role in insulin-induced epithelial-mesenchymal transition in breast cancer cells. BMC Cancer, 20, 626.

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Adipogenic Differentiation of SVF Cells

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SVF and adipocyte progenitor cells were isolated as described above. Cell pellets were resuspended in DMEM with 10% FBS and then filtered through a 40 μm cell strainer. Stroma vascular cells were then used for cell culture. Adipogenesis was induced by culturing 100% confluent cells in DMEM containing 10% FBS (GemCell), 0.5 mM 3-isobutyl-1-methylxanthine (Sigma), 1mM dexamethasone (Sigma) and 5 μg/ml insulin (Sigma) for 2 days and then the cell culture was changed to maintenance media containing 5 μg/ml insulin for 6 days. Minimal differentiation stimulation was with 5 μg/ml of insulin (Sigma).

Chen M., Kim S., Li L., Chattopadhyay S., Rando T.A, & Feldman B.J. (2023). Identification of an adipose tissue-resident pro-preadipocyte population. Cell reports, 42(5), 112440.

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Isolation and Decidualization of Human Endometrial Stromal Cells

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Isolation of human endometrial stromal cells was carried out as described previously.⁸ Endometrial stromal cells were seeded to 6‐well Costar plates (Sigma‐Aldrich) at a density of 10⁵/well and cultured in DMEM/F12‐Glutamax medium (Thermo Fischer Scientific) supplemented with 10% heat‐inactivated foetal bovine serum (HI‐FBS; Sigma‐Aldrich) and 0.2% penicillin‐streptomycin (Sigma‐Aldrich) until 80%–90% confluency. In vitro decidualization of endometrial stromal cells was induced in phenol red‐free DMEM/F12 (Thermo Fischer Scientific) supplemented with 2% charcoal‐stripped FBS (Sigma‐Aldrich) 0.2% penicillin‐streptomycin (Sigma‐Aldrich) using 1 μM medroxyprogesterone‐17‐acetate (MPA; Sigma‐Aldrich) and 0.5 mM dibutyryl‐cAMP (db‐cAMP; Sigma‐Aldrich) in the presence or absence of 100 nM insulin (Sigma‐Aldrich), 1 μM DHT (Sigma‐Aldrich) or 1 μM testosterone (Sigma‐Aldrich), or the combination of insulin and DHT or insulin and testosterone for six days. The culture media was renewed after 3 days.
In the experiments of flow cytometry, wound‐healing assay and spheroid co‐culture invasion assay, we chose to treat the cells with insulin and/or DHT, but not with testosterone, since DHT has the strongest affinity to the androgen receptor²¹ and does not convert to other hormones.

Hirschberg A.L., Jakson I., Graells Brugalla C., Salamon D, & Ujvari D. (2021). Interaction between insulin and androgen signalling in decidualization, cell migration and trophoblast invasion in vitro. Journal of Cellular and Molecular Medicine, 25(20), 9523-9532.

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Adipogenic Induction Protocol for C2C12 and NIH3T3 Cells

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Murine C2C12 myoblasts [74 (link), 75 (link)] and NIH3T3 fibroblasts [76 (link)] were cultured in 24-well plates at a cell density of 12,000 and 30,000 cells per well, respectively. Both cell lines were incubated at 37°C/5% CO₂, using a DMEM-based growth medium containing 10% FBS.
When indicated, cells were switched to adipogenic induction conditions (adapted from [77 (link)]). Briefly, near-confluent cultures were switched into DMEM-based medium containing 10% FBS, 0.5 mM isobutylmethylxanthine, 1 μg/ml dexamethasone, and 5 μg/ml insulin (Sigma-Aldrich)] for two days followed by a switch to a high-insulin medium consisting of DMEM with 10% FBS and 5 μg/ml insulin for the remainder of the culture time. The high insulin medium was typically changed every other day or daily when reaching high density. For control cultures, a medium change to fresh standard growth medium was applied at the time the parallel cultures were switched to adipogenic induction conditions and at any subsequent medium change.

Phelps M., Stuelsatz P, & Yablonka-Reuveni Z. (2016). Expression profile and overexpression outcome indicate a role for βKlotho in skeletal muscle fibro/adipogenesis. The FEBS journal, 283(9), 1653-1668.

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Adipose-Derived Stromal Cell Differentiation

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The stromal vascular faction (SVF) cells derived from iWAT were obtained as previously described.^{38 (link)} SVF cells were cultured in DMEM/F12 (Corning, 10-090-CV) containing 10% FBS (GenClone, 25-514), 1% penicillin/streptomycin (Gibco, 10378016), 20 nM insulin (Sigma), and 1 nM triiodothyronine (T3, Sigma, T6397). Two days after becoming confluent (defined as Day 0), SVF cells were induced with DMEM/F12 containing 10% FBS, 1% penicillin/streptomycin, 0.5 mM isobutylmethylxanthine (IBMX, Sigma, I7018), 125 μM indomethacin (Sigma, I7378), 1 μM dexamethasone (Sigma, D4902), 20 nM insulin, and 1 nM T3 for 48 h. Cells were maintained in DMEM/F12 containing 10% FBS, 1% penicillin/streptomycin, 20 nM insulin, and 1 nM T3 until lipid drop appeared. This medium was replenished every two days.

Zhang Z., Huang Z., Ong B., Sahu C., Zeng H, & Ruan H.B. (2019). Bone marrow adipose tissue-derived stem cell factor mediates metabolic regulation of hematopoiesis. Haematologica, 104(9), 1731-1743.

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Adipocyte Differentiation Induction Protocol

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Following cell confluence, the medium for inducing differentiation was used, and changed every 2 days until day 5 of differentiation. Detailed procedures for the different treatments were described as in Figure 1. The basal medium was prepared using DMEM/F12 and 10% FBS. IDX was composed of 0.25 μM dexamethasone (Sigma), 10 μg/ml insulin (Sigma) and 0.5 mM IBMX (Sigma). Cells in the control group were cultured in the basal medium from 0 to 120 h. Cells in the oleate (Sigma) group were treated in the basal medium supplemented with oleate from 0 to 120 h. Cells in the IDX group were cultured in the basal medium supplemented with insulin, dexamethasone and IBMX at 0 h, and switched to insulin alone after 48 h, which was similar to mouse 3T3-L1 cells [19 (link)]. Cells in the IDX plus oleate group were cultured in the basal medium supplemented with insulin, dexamethasone, IBMX and oleate at 0 h, and switched to insulin and oleate after 48 h. The final concentration of oleate in the oleate group and the IDX plus oleate group was 160 μM.

Shang Z., Guo L., Wang N., Shi H., Wang Y, & Li H. (2014). Oleate promotes differentiation of chicken primary preadipocytes in vitro. Bioscience Reports, 34(1), e00093.

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Adipogenic Differentiation of SVF Cells

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Chen M., Kim S., Li L., Chattopadhyay S., Rando T.A, & Feldman B.J. (2023). Identification of an adipose tissue-resident pro-preadipocyte population. Cell reports, 42(5), 112440.

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Breast Cancer Cell Line Cultivation

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BT474, BT549, Hs578T, MCF7, MDA-MB-231, MDA-MB-468, SK-BR-3, and T-47D, cells were purchased from the American Type Culture Collection (ATCC). HMLE cells were a kind gift from Robert Weinberg, Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology. BT474, BT549, MDA-MB-231, MDA-MB-468, SK-BR-3, and T-47D were cultured in RPMI 1640 (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (Biochrom) and 1% penicillin-streptomycin (Sigma-Aldrich). BT549 cells were grown in the presence of 0.001 mg/ml insulin (Sigma-Aldrich) and T-47D were grown in the presence of 0.006 mg/ml insulin (Sigma-Aldrich). Hs578T were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma-Aldrich) supplemented with 10% FBS, 1% penicillin-streptomycin, and 0.01 mg/ml insulin. MCF7 were cultivated in Minimum Essential Medium Eagle (MEM; Sigma-Aldrich) supplemented with 10% FBS, 1% penicillin-streptomycin, and 0.01 mg/ml insulin. HMLE cells were grown in a 1:1 mixture of MEBM (Lonza) with DMEM/F12 (Sigma-Aldrich) supplemented with 10 ng/ml EGF, 0.5 μg/ml hydrocortisone, 0.01 mg/ml insulin, and 1% penicillin-streptomycin. All cell lines were incubated in a 5% CO2 humidified incubator at 37°C.

Tellez-Gabriel M., Tekpli X., Reine T.M., Hegge B., Nielsen S.R., Chen M., Moi L., Normann L.S., Busund L.T., Calin G.A., Mælandsmo G.M., Perander M., Theocharis A.D., Kolset S.O, & Knutsen E. (2022). Serglycin Is Involved in TGF-β Induced Epithelial-Mesenchymal Transition and Is Highly Expressed by Immune Cells in Breast Cancer Tissue. Frontiers in Oncology, 12, 868868.

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