To generate erythroid cells, sorted CD34+ progenitors were plated in 0.1% gelatin-coated plates (1.0×106/well in a 6-well plate) and cultured for 4 days in erythroblast induction medium, which consisted of 49% IMDM (Life Technologies, 12440), 49% Ham’s F12 (Life Technologies, 11765), 1% ITSX, 1% lipid (Life Technologies, 11905-031), 5 mg/100 mL I-ascorbic acid (Sigma, A8960), 500 mg/100 mL BSA (Sigma, A7030), and 200uM 1-thioglycerol (Sigma, M6145), supplemented with 100 ng/mL SCF (PeproTech, 300–07), 10 ng/mL interleukin-3 (PeproTech, 200–03), 5 U/mL EPO (R&D Systems, 287-TC), 40 ng/mL insulin-like growth factor-1(Sigma I-3769), 1 μM Dexamethsone (Sigma, D2915), and 50 ng/mL Flt3-L (PeproTech, 300–19) (Figure 1 A). Unlike adherent ECs, induced erythroblasts detached on generation and were collected as nonadherent cell fraction at day 4 for characterization.
Insulin like growth factor 1
Manufactured by Merck Group
Sourced in United Kingdom, United States
Insulin-like growth factor-1 (IGF-1) is a protein that plays a crucial role in cell growth and development. It is a key regulator of the body's response to growth hormone and is involved in a variety of physiological processes, including cell proliferation, differentiation, and metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Rapamycin, by Merck Group (2 mentions)
Ham s f12, by Thermo Fisher Scientific (1 mentions)
Flt3l, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Stem cell factor (scf), by Thermo Fisher Scientific (1 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (1 mentions)
Interleukin 3 (il 3), by Thermo Fisher Scientific (1 mentions)
Its x, by Thermo Fisher Scientific (1 mentions)
1 thioglycerol, by Merck Group (1 mentions)
Dexamethsone, by Merck Group (1 mentions)
Fibrinogen, by Merck Group (1 mentions)
Thrombin, by Merck Group (1 mentions)
Aminocaproic acid, by Merck Group (1 mentions)
Quanta 450 feg, by Thermo Fisher Scientific (1 mentions)
Sunset yellow fcf, by Merck Group (1 mentions)
Lithium phenyl 2 4 6 trimethylbenzoylphosphinate, by Merck Group (1 mentions)
Anti tra 1 60, by Merck Group (1 mentions)
Bq123, by Merck Group (1 mentions)
Alexa fluor 488 chicken anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 goat anti mouse igg1, by Thermo Fisher Scientific (1 mentions)
9 protocols using insulin like growth factor 1
1
Erythroblast Induction from CD34+ Progenitors
2
Fabrication of 3D Skeletal Muscle Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fabrication of 3D skeletal muscle and hydrogel structure was described previously in detail. Briefly, the cell‐matrix solution consisted of 1 × 107 cells/mL of MPCs, 30% (vol/vol) Matrigel, 4 mg/mL fibrinogen (Sigma‐Aldrich), and 0.5 U of thrombin (Sigma‐Aldrich)/mg of fibrinogen was seeded in a polydimethylsiloxane ring mould with 5 mm/6.6 mm for inner/outer diameters. After 2 h, the PM supplemented with 1 mg/mL aminocaproic acid (Sigma‐Aldrich) was added to the ring mould and changed every other day. After 7 days, compacted muscle rings were transferred into the 3D‐printed hydrogel cantilevers and cultured with the DM supplemented with 1 mg/mL of aminocaproic acid and 0.5 ng/mL of insulin‐like growth factor‐1 (Sigma‐Aldrich). The 3D hydrogel cantilever structure was designed in Solidworks (Figure S1 ) and fabricated using the digital light processing 3D printer (PICO2, Asiga) and a printing resin solution containing 20% (vol/vol) polyethylene glycol diacrylate M.W. 700 (Sigma‐Aldrich), 1 mg/mL of lithium phenyl‐2,4,6‐trimethylbenzoylphosphinate (Sigma‐Aldrich), and 0.4 mg/mL of Sunset Yellow FCF (Sigma‐Aldrich). Myofibre alignment of the 3D muscle ring was confirmed by scanning electron microscopy (FEI Quanta FEG 450).
3
Immunofluorescence Staining of Pluripotency and Cardiac Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence staining for pluripotency of iPSCs was performed by using the following primary antibodies: anti‐NANOG (Abcam), anti‐OCT3/4 (Santa Cruz), anti–SSEA 3 (Millipore), anti–SSEA 4 (Millipore), anti–Tra‐1‐60 (Millipore), and anti–Tra‐1‐81 (Millipore). In immunofluorescence staining for cardiac markers, monoclonal anti–α‐actinin (Sigma), monoclonal anti‐cTnT (Thermo Scientific), polyclonal anti‐cTnT (Santa Cruz), monoclonal anti–myosin light chain (MLC)2a (Synaptic Systems), polyclonal anti‐MLC2v (ProteinTech Group), anti‐ANP (Santa Cruz), anti–cMyBP‐C (Santa Cruz), polyclonal anti–cMyBP‐C motif (supplied by C. Witt University of Heidelberg, Heidelberg, Germany), and anti–nuclear factor of activated T cells (NFAT)c4 (Santa Cruz) were used. The isotype‐specific secondary antibodies, Alexa Fluor 488 chicken anti‐rabbit IgG, Alexa Fluor 594 goat anti‐mouse IgG1, Alexa Fluor 488 goat anti‐rat IgM, Alexa Fluor 594 goat anti‐mouse IgM, Alexa Fluor 488 goat anti‐mouse IgG, Alexa Fluor 594 goat anti‐mouse IgG2b, Alexa Fluor 594 chicken anti‐goat IgG, and Alexa Fluor 555 goat anti‐rabbit IgG, were all obtained from Invitrogen. The tested drugs included endothelin‐1 (1.0, 10, 100, or 1000 nmol/L), angiotensin II (100 nmol/L), insulin‐like growth factor 1 (100 nmol/L), phenylephrine (0.05 mmol/L), BQ‐123 (250 nmol/L), and BQ‐788 (100 nmol/L) (all from Sigma).
4
Pharmacological Interventions in Cell Modeling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The drugs employed in this study were FTI-277, Pravastatin, Zoledronic acid, Rapamycin, Insulin-like growth factor 1, N-acetyl-l -cysteine and GGTI-2133 and were all obtained from Sigma Aldrich, UK. Drugs were added to the media and incubated for fixed periods of time. The final concentration and duration of drug treatments were: FTI-277—2.5 µM for 48 h (Mehta et al. 2011 (link)), Pravastatin—1 µM for 24 h (Varela et al. 2008 (link)), Zoledronic acid—1 µM for 24 h, Rapamycin—10 nM for 24 h (Cao et al. 2011b ), Insulin-like growth factor 1—50.0 ng/mL for 24 h (Mariño et al. 2010 (link)), N-acetyl-l -cysteine—20 µM for 1 h (Richards et al. 2011 (link)), FTI-277 and GGTI-2133—both 2.5 µM for 48 h) (Mehta et al. 2011 (link); Kieran et al. 2007 (link)), Pravastatin and Zoledronic acid—both 1 µM for 24 h (Varela et al. 2008 (link)) and FTI-277—2.5 µM for 48 h, Pravastatin and Zoledronic acid—both 1 µM for 24 h.
5
Ovarian Cancer Cell Line Culturing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
OVCAR-3, SKOV-3, and Caov-3 were purchased from the American Type Culture Collection (Manassas, VA, USA). COV434 cells were obtained from the European Collection of Authenticated Cell Cultures (ECACC, Sigma-Aldrich, St. Louis, MO, USA). The human ovarian serous carcinoma cell lines OVCAR-3, SKOV-3, and Caov-3 were cultured in RPMI 1640, McCoy’s, and Dulbecco’s modified Eagle’s medium (DMEM), respectively. Media were supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific Inc., Carlsbad, CA, USA). The human metastatic granulosa cell line COV434 was cultured in DMEM supplemented with 2 mM l -glutamine (Biowest, Nuaillé, France) and 10% FBS. All cell cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2.
Human adiponectin, 17β-estradiol, progesterone, and insulin-like growth factor 1 were purchased from Sigma-Aldrich. BPA (AccuStandard, New Haven, CT, USA) and E2 were dissolved in absolute ethanol. TBBPA and TCBPA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and dissolved in DMSO. The final concentration of ethanol and DMSO in the medium was 0.1%. Viability of cells was not affected at this concentration.
Human adiponectin, 17β-estradiol, progesterone, and insulin-like growth factor 1 were purchased from Sigma-Aldrich. BPA (AccuStandard, New Haven, CT, USA) and E2 were dissolved in absolute ethanol. TBBPA and TCBPA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and dissolved in DMSO. The final concentration of ethanol and DMSO in the medium was 0.1%. Viability of cells was not affected at this concentration.
6
Extracellular and Intracellular Solutions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In most experiments, cells were kept in extracellular solution containing (mM) 144 NaCl, 5.8 KCl, 0.9 MgCl2, 1.3 CaCl2, 0.7 NaH2PO4, 5.6 D-glucose, and 10 HEPES, pH 7.4 (with NaOH), 305–310 mosm/kg. Solutions with increased K+ concentration were made by replacing NaCl with KCl. For maximal depolarization, a 150 mM K+ solution was required prepared as follows (mM): 150 KCl, 0.9 MgCl2, 1.3 CaCl2, 0.7 NaH2PO4, 5.6 D-glucose, and 10 HEPES, pH 7.4 (with KOH). For the TRPV1 and ChR2(H134R) experiments Ca2+ was omitted from the standard extracellular solution to prevent the Ca2+ dependent activation of phospholipases-C (PLC) (Ca2+-free extracellular solution) (Rebecchi and Pentyala, 2000 (link)). Intracellular solution used for patch clamp experiments contained (mM) 135 KCl, 2.41 CaCl2, 2.5 MgCl2, 5 EGTA, 5 HEPES, and 3 Na2-ATP, pH 7.3 (with KOH), 290–295 mosm/kg.
Capsaicin and insulin-like growth factor-1 were purchased from Sigma-Aldrich, the bisperoxovanadate from Santa Cruz Biotechnology (Dallas, TX), DTT from Fermentas (St. Leon-Rot, Germany).
Capsaicin and insulin-like growth factor-1 were purchased from Sigma-Aldrich, the bisperoxovanadate from Santa Cruz Biotechnology (Dallas, TX), DTT from Fermentas (St. Leon-Rot, Germany).
7
Cell Line-Based Phosphoinositide Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CHO and HEK 293 cells were plated on glass coverslips or—for TIRF experiments—glass bottom dishes (WillCo Wells B. V., Amsterdam, The Netherlands). For HEK 293 cells the glass was coated with poly-D-lysine. Cells were transfected 24–48 h later using JetPEI (Polyplus Transfection, Illkirch, France) as described (Halaszovich et al., 2009 (link)). For the used expression vectors see Supplementary Table S1 . Bovine phosphatidylinositol 3-kinase p110α (constitutively active mutant K227E) was cotransfected with PTENCiV to increase PI(3,4,5)P3 levels except for experiments involving AktAR (Rodriguez-Viciana et al., 1996 (link)). In the latter experiments, PI(3,4,5)P3 levels were elevated by incubating the cells with 0.4 ng/μl insulin-like growth factor-1 (Sigma, St.Louis, MO) for 15 min (Halaszovich et al., 2009 (link)). HEK cells were preferred for confocal imaging, since they present a stronger membrane localization of PI markers. Experiments were performed at room temperature 24–48 h after transfection.
8
Osteosarcoma Cell Lines and IGF-1 Activation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
U2OS and Saos-2 are the cells lines of human osteosarcoma which were purchased from Sigma Aldrich, USA. U2OS and Saos-2 cells were cultured in IMDM (Iscove's modified Dulbecco's medium) with 100 U/ml benzyl penicillin: 100 μg/ml streptomycin solution and 10% (v/v) inactivated fetal bovine serum. The temperature for the solution was maintained at 37°C in a humidified incubator with 5% CO2. Insulin-like growth factor-1 was obtained from Sigma Aldrich, USA. 25 ng/ml IGF-1 was added to the media along with desflurane, isoflurane, and sevoflurane treatments, respectively, for activation of the PI3K/AKT pathway.
9
Investigating PI3Kγ Signaling Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human SDF1α was purchased from PeproTech; UK14304, GW5074, rapamycin, BFA, ilimaquinone, monensin, nigericin, swainsonine, and LY294002 were from Sigma Aldrich; nocodazole, insulin-like growth factor 1, AMD3100, control siRNA (medium GC), siRNAs targeting to human PI3Kγ regulatory subunits p101 and p87, and antibodies against GFP, phospho-ERK1/2, Gγ9, and β-actin and the PI3Kγ subunits p110γ, p101 and p87 were from Santa Cruz Biotechnology; wortmannin, AS-604850, and GSK2292767 were from ApexBio; TGX-221 and HS-173 were from Adooq Bioscience; U0126 and PD98059 were from Calbiochem; EGF, puromycin, and blasticidin S were from Thermo Fisher Scientific; U-73122 and AZD5363 were from MedChemExpress; U-73433, CRT0066101, and Go6976 were from Cayman Chemical; PTX was from List Biological Laboratories; gallein and rauwolscine were from Tocris Bioscience; D-Luciferin was from GoldBio; antibodies against hemagglutinin and ERK1/2 were from Cell Signaling Technology; antibodies against Gγ3 were from Abcam. All other materials were obtained as described (52 (link), 53 (link)).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!