WT/WT and ΔE9/ΔE9 hiPSCs were differentiated into MGLs using a previously described protocol (McQuade et al., 2018 (link)). Briefly, hiPSCs were differentiated into hematopoietic progenitor cells (HPCs) using the STEMdiff™ Hematopoietic Kit (#05310, Stem cell Technologies). HPCs were either frozen using Bambanker HRM freezing media (#BBH01; Bulldog Bio) or further differentiated into MGLs. HPCs were cultured in microglia differentiation medium comprised of DMEM/F12 (#11039047; Thermo Fisher Scientific), B27 (#17504-044; Thermo Fisher Scientific), N2 (#17502-048; Thermo Fisher Scientific), insulin-transferrin-selenite (#41400045; Thermo Fisher Scientific), non-essential amino acids (#11140050; Thermo Fisher Scientific), Glutamax (#35050061; Thermo Fisher Scientific), human insulin (#I2643-25 mg; Sigma) and monothioglycerol (#M1753; Sigma) supplemented with 25 ng/ml human M-CSF (#PHC9501; Thermo Fisher Scientific), 50 ng/ml TGF-β1 (#130-108-969; Miltenyl), and 100 ng/ml IL-34 (#200-34; Peprotech). After 24 days in this medium, two additional cytokines, 100 ng/ml CD200 (#C311; NovoProtein), and 100 ng/ml CX3CL1 (#300-31; PeproTech), were added to the medium described above to mature MGLs. MGLs were cultured in this new medium for an additional week and then harvested for experiments.
Insulin transferrin selenite
Manufactured by Thermo Fisher Scientific
Sourced in United States
Insulin-transferrin-selenite is a laboratory supplement that contains a combination of insulin, transferrin, and sodium selenite. It is commonly used to support cell growth and proliferation in cell culture media.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (3 mentions)
Dmem f12, by Thermo Fisher Scientific (3 mentions)
Hydrocortisone, by Merck Group (3 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Human insulin, by Merck Group (1 mentions)
Monothioglycerol, by Merck Group (1 mentions)
Il 34, by Thermo Fisher Scientific (1 mentions)
Cd200, by Novoprotein (1 mentions)
Cx3cl1, by Thermo Fisher Scientific (1 mentions)
Stemdiff hematopoietic kit, by STEMCELL (1 mentions)
Mdv3100, by Selleck Chemicals (1 mentions)
Estradiol, by Merck Group (1 mentions)
Tms 013 bkr, by Merck Group (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Matrigel coated, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium f12, by Thermo Fisher Scientific (1 mentions)
Accutase, by Merck Group (1 mentions)
9 protocols using insulin transferrin selenite
1
Differentiation of hiPSCs into Microglia
2
Prostate Cancer Cell Lines and AR Inhibition
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We obtained cell lines from ATCC, including AR-positive PCa cell lines (LNCaP and C4-2), AR-independent PCa cell lines (PC3 and LASCPC-01), and normal prostate epithelial cell line (PZ-HPV-7). LNCaP, C4-2, and PC3 cells were cultured in RPMI-1640 medium (ThermoFisher Scientific, 11875-085, New York, NY, USA) containing 5% fetal bovine serum (FBS; EMD Millipore, TMS-013-BKR, Billerica, MA, USA) and 1% penicillin. NEPC-like LASCPC-01 cells were cultured in RPMI-1640 medium supplemented with 10 nM hydrocortisone (Sigma-Aldrich, H0888, Burlington, NJ, USA), one vial insulin/transferrin/selenite (ThermoFisher Scientific, 41400-045), 200 nM-estradiol (Sigma-Aldrich, E2758, Burlington, NJ, USA), 5% FBS, and 1% penicillin. PZ-HPV-7 cells were cultured in keratinocyte serum-free medium (K-SFM; ThermoFisher, 17005-042) containing 0.05 mg/mL bovine pituitary extract (BPE; ThermoFisher) and 5 ng/mL human recombinant epidermal growth factor (EGF; ThermoFisher). Six months of AR inhibition was performed using an AR antagonist with 20 µM enzalutamide (MDV3100; Selleckchem, S1250, Houston, TX, USA).
3
hESC Culture and Maintenance Protocol
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Experiments were done with the hESC line (RH6) (59 (link)). hESCs were grown on a feeder layer of mouse embryonic fibroblast cells, which were inactivated with 10 μg/ml mitomycin C. hESCs were fed daily with Dulbecco's modified Eagle's medium/F-12 (Gibco; catalog no.: 21331-020) complemented with 20% knockout serum replacement (Gibco; catalog no.: 10828-028), 1% nonessential amino acids (Gibco; catalog no.: 11140-035), 0.1 mM β-mercaptoethanol (Sigma–Aldrich; catalog no.: M3148), 1% antibiotic mixture comprising 100 units/ml penicillin and 100 μg/ml streptomycin (Gibco; catalog no.: 15070063), insulin–transferrin–selenite (Gibco; catalog no.: 41400-045), 2 mM l -glutamine (Gibco; catalog no.: 25030-024), and 100 ng/ml basic fibroblast growth factor (Royan Institute) in an ideal atmosphere of 37 °C with 5% CO2 air-humidified incubator. The growth medium was treated daily with a 10 μM ROCK inhibitor (Y-27632; Calbiochem; catalog no.: 688000), and hESC colonies were passaged by collagenase IV (Gibco; catalog no.: 17104-019) in Dulbecco's modified Eagle's medium/F12 for 7 min. For dissociation into a single cell, hESCs were washed with PBS− and then treated with accutase (Millipore; catalog no.: SCR005) at 37 °C for 3 min and harvested by pipetting, then cultured to Matrigel-coated (Sigma–Aldrich; catalog no.: E1270) dishes.
4
EMT Regulation by AhR Agonists
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Ham’s F12 medium, Dulbecco’s Modified Eagle’s Medium (DMEM) (high glucose), fetal-bovine serum (FBS; heat inactivated), L-glutamine, HEPES, insulin-transferrin-selenite (ITS), estrogen, hydrocortisone, penicillin-streptomycin solution, and sodium pyruvate solution was obtained from Gibco (Gaithersburg, MD, USA). The AhR agonists, TCDD and CH223191, were obtained from Sigma-Aldrich (St. Louis, MO, USA). IP was purchased from Supelco® and FICZ was obtained from Enzo Life Sciences, Inc., Farmingdale, NY, USA. Gelatin from porcine skin and casein was purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA). For western blotting, the following antibodies were utilized: anti-E-cadherin (#3195), β-catenin (#8480), anti-rabbit IgG–HRP (#7074) and anti-mouse IgG–HRP (#7076). All of these compounds were purchased from Cell Signalling (Cell Signalling Technology, Danvers, MA, USA), while anti-Vimentin (GTX00619) and anti-GAPDH (GTX100118) were purchased from Gene Tex (GeneTex, Irvine, CA, USA). Anti-Fibronectin (AB1954-25UL) was obtained from Merck (Darmstadt, Germany).
5
Hypoxia-Reoxygenation in Proximal Tubular Cells
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Human proximal tubular epithelial cells (TECs) (HK-2 cell line; CRL-2190; ATCC, VA) were maintained in RPMI 1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 1% insulin transferrin selenite (Gibco) and 5 ng/ml hydrocortisone (Sigma-Aldrich, St. Louis, MO, USA). Cells were cultured at 0.25x106 cells/ml in serum-free medium for 24 h before the assay and further stimulated with thapsigargin (Tg; 4 μM; 24 h; Sigma-Aldrich). Inhibitors used were: JQ1(+) or its inactive enantiomer JQ1(-) (100-500 nM; 24 h; Selleckchem, Houston, TX, USA); I-BET 726 (400-1000 nM; 24 h; Cayman Chemical, Ann Arbor, MI, USA) and 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside DRB (20-60 μM; 6 h; Sigma-Aldrich). For hypoxic conditions, cells were cultured in medium without nutrients for 12 h in a hypoxic incubator (1% O2, 94% N2 and 5% CO2, 37°C). In some conditions, HK-2 cells were followed by reoxygenation at different times (2, 4, and 6 h) with fresh complete medium, and in 5% CO2 and 95% air at 37°C.
6
Cardiosphere Formation of c-Kit+ CPCs
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c-Kit+ CPCs were incubated in Dulbecco’s MEM and Ham’s F12 (ratio 1:1; Sigma), bFGF (10 ng/ml), EGF (20 ng/ml), LIF (10 ng/ml), insulin-transferrin-selenite (Gibco), 1x B27 (Gibco), 1x N2 (Gibco), 1% penicillin-streptomycin, 1% fungizone, and gentamicin in the presence or absence of SP at the indicated concentration [30 (link)]. After 2 weeks, the cardiosphere formation of c-Kit+ CPCs was visualized with an Olympus BX51 microscope equipped with a 20x lens. The number of spheres was counted manually from brightfield images using the ImageJ cell counter plugin and expressed as a percentage. All bright-field images were selected with clone identities blinded and at least 20 random images were obtained from each well.
7
Expansion of Human Embryonic Stem Cells as Spheroids
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In this experimental study, hESCs (RH5 line) were
cultured and expanded as spheroids according to a
previously described protocol (22 (link)). Briefly, 2×105
viable cells/ml were cultured in hESC medium that
consisted of Dulbecco’s Modified Eagle Medium/
Ham’s F-12 (DMEM/F12, Gibco, USA) supplemented
with 20% knockout serum replacement (KOSR, Gibco,
USA), 1% insulin-transferrin-selenite (Gibco, USA),
1% nonessential amino-acids (NEAA, Gibco, USA),
1% penicillin/streptomycin (Gibco, USA), 0.1 mM
ß-mercaptoethanol (Sigma-Aldrich, USA), and 100 ng/
ml basic fibroblast growth factor (bFGF, Royan Biotech,
Iran) in non-adhesive bacterial plates. The medium was
renewed every 2 days. When spheroids reached 200-250
µm, they were dissociated into single cells with Accutase
solution (Sigma-Aldrich, USA), and replated on new
bacterial plates at a 1:3 ratio. Cells were treated with 10
µM of ROCK inhibitor (ROCKi, Sigma-Aldrich, USA)
for the first 2 days.
cultured and expanded as spheroids according to a
previously described protocol (22 (link)). Briefly, 2×105
viable cells/ml were cultured in hESC medium that
consisted of Dulbecco’s Modified Eagle Medium/
Ham’s F-12 (DMEM/F12, Gibco, USA) supplemented
with 20% knockout serum replacement (KOSR, Gibco,
USA), 1% insulin-transferrin-selenite (Gibco, USA),
1% nonessential amino-acids (NEAA, Gibco, USA),
1% penicillin/streptomycin (Gibco, USA), 0.1 mM
ß-mercaptoethanol (Sigma-Aldrich, USA), and 100 ng/
ml basic fibroblast growth factor (bFGF, Royan Biotech,
Iran) in non-adhesive bacterial plates. The medium was
renewed every 2 days. When spheroids reached 200-250
µm, they were dissociated into single cells with Accutase
solution (Sigma-Aldrich, USA), and replated on new
bacterial plates at a 1:3 ratio. Cells were treated with 10
µM of ROCK inhibitor (ROCKi, Sigma-Aldrich, USA)
for the first 2 days.
8
Osteoclastogenic Signaling Pathway Regulation
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Jtg (Purity > 95%) was obtained from Ginwa (Xi’an, China), and dissolved in Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Milwaukee, WI, United States). α-minimum essential medium (α-MEM) and Dulbecco’s minimum essential medium (DMEM) were purchased from Hyclone (Logan, UT, United States). Fetal bovine serum, insulin-transferrin-selenite (Livshits et al., 2010 (link)) and penicillin/streptomycin were purchased from Gibco (New York, NY, United States). Receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony stimulating factor (M-CSF) were obtained from R&D Systems (Minneapolis, MN, United States). H2O2 solution, the TUNEL apoptosis detection kit, cell counting kit-8 assay, DCFH-DA probe and RIPA lysis buffer were obtained from Beyotime Biotechnology (Shanghai, China). Antibodies against nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) were purchased from Cell Signaling Technology (Danvers, MA, United States). Rhodamine-conjugated phalloidin, 4′,6-diamidino-2-phenylindole (DAPI) and TRIzol reagent were obtained from Invitrogen (Carlsbad, CA, United States).
9
Optimizing Endothelial Differentiation from Bone Marrow
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Endothelial differentiation culture medium (EDCM) was optimized using unsorted BM. EDCM contained Iscove's Modified Dulbecco's Medium (Gibco, Carlsbad, CA, https://www.thermofisher.com/us/en/home/brands/gibco.html ), and supplementary factors: 20% fetal calf serum (FCS) (Thermo Scientific, Waltham, MA, https://www.thermofisher.com/se/en/home.html ), 2 mM l ‐glutamine, 1% insulin‐transferrin‐selenite, 1% nonessential aminoacids, 0.5% penicillin‐streptomycin, 0.1% β‐mercaptoethanol (Gibco), 15 IU/ml heparin (Fisons Pharmaceuticals, Ipswich, U.K.), 50 ng/ml vascular endothelial growth factor (Sigma Aldrich, MO, http://www.sigmaaldrich.com ), 75 µM ascorbic acid (Sigma Aldrich), 1 µM hydrocortisone (Sigma Aldrich), and 5 ng/ml basic fibroblast growth factor (Sigma Aldrich). For plate‐adherent cell isolation, dulbecco modified eagle medium (DMEM) with supplementary factors: 20% FCS, 2 mM l ‐glutamine, 1% insulin‐transferrin‐selenite, 1% non‐essential amino acids and 0.5% penicillin‐streptomycin was used (all reagents from Gibco). Long‐term culture growth medium contained DMEM, 10% FCS, 1,000 U/ml leukemia inhibitory factor, 10 ng/ml epidermal growth factor, and 10 ng/ml platelet‐derived growth factor (Sigma Aldrich).
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