Human podocytes were kindly provided by Prof. Saleem and maintained in RPMI 1640 medium supplemented with 10% foetal bovine serum (FBS; F7524, Sigma-Aldrich, St. Louis, MO, USA), Insulin-Transferrin-Selenium supplement (ITS; Sigma-Aldrich, St. Louis, MO, USA), 2 mM L-glutamine, and antibiotic mixture, as previously reported [35 (link)]. To stimulate cell proliferation, podocytes were cultivated at 33 °C in 5% CO2 (permissive conditions). To induce differentiation, they were maintained at 37 °C in 5% CO2 (non-permissive conditions) for at least 2 weeks, and verified to be free of mycoplasma contamination through the N-GARDE Mycoplasma PCR Detection kit (EuroClone S.p.A, Milan, Italy). Cell density was kept below 90% to allow differentiation. Cells were used between passages 7 and 12. To test albumin effects, cells were cultured in serum deprivation (1%) starting from 24 h before stimulation.
Insulin transferrin selenium supplement
Manufactured by Merck Group
Sourced in United States, United Kingdom
Insulin-transferrin-selenium supplement is a lab equipment product that provides a combination of insulin, transferrin, and selenium. This supplement is designed to support cell growth and proliferation in cell culture environments.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (2 mentions)
F7524, by Merck Group (1 mentions)
Indomethacin, by Merck Group (1 mentions)
Isobutylmethylxanthin, by Merck Group (1 mentions)
Oil red o, by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Penicillin and streptomycin, by Roche (1 mentions)
Nonessential amino acid solution, by Thermo Fisher Scientific (1 mentions)
Fatty acid free bsa, by Merck Group (1 mentions)
Matrigel, by BD (1 mentions)
Dmem f12 medium, by Corning (1 mentions)
Transwell filter inserts, by BD (1 mentions)
Triiodothyronine, by Merck Group (1 mentions)
Dmem ham s f 12, by American Type Culture Collection (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
9 protocols using insulin transferrin selenium supplement
1
Differentiation and Maintenance of Human Podocytes
2
Adipogenic Differentiation of MSCs
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MSCs were seeded at 20,000 cells/cm2 into a 24-well plate. Cells were then cultured in Adipogenic medium (10% FBS; 10% insulin-transferrin-selenium supplement, Sigma-Aldrich, Gillingham, UK; 10−8 M dexamethasone, Sigma-Aldrich, Gillingham, UK; 0.5 mM isobutylmethylxanthin, Sigma-Aldrich; 100 µM indomethacin, Sigma-Aldrich, Gillingham, UK). The media was changed every 2 days and after 14 days cells were analysed by Oil Red O (Sigma-Aldrich, Gillingham, UK) staining and photos were taken.
3
Immortalized Human Podocyte Cell Culture
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The immortalized human podocyte cell line was obtained from Dr Saleem (48 (link)) and cultured in RPMI 1640–based medium supplemented with 10% fetal bovine serum (Invitrogen), 2 g/l of sodium bicarbonate (NaHCO3), insulin-transferrin-selenium supplement (Sigma-Aldrich), and 200 units/ml penicillin and streptomycin (Roche Applied Science) as described (6 (link), 49 (link)). HEK293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen) and 200 U/ml of penicillin and streptomycin (Invitrogen). Lipofectamine 2000 (Invitrogen) was used to perform transfection according to the manufacturer's protocol. HEK293 cells overexpressing FLAG NEPH1/NEPHRIN were plated and grown to 90% confluency and then serum starved for 2 h. Cells were then treated with fresh pervanadate (1 mM) for 45 min at 37 °C as described (50 (link)). Vehicle-treated cells were used as controls. After pervanadate treatment, cells were lysed in RIPA buffer supplemented with phosphatase and protease inhibitors. Protein estimation from each lysate was performed using the BCA protein kit, and equivalent amounts of lysates were subjected to SDS-PAGE and immunoblot analysis.
4
Immortalized Human Podocyte Cell Line
5
Bovine In Vitro Embryo Culture
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The culture medium was composed of modified synthetic oviduct fluid (mSOF) [21 (link), 22 (link)] supplemented with 20 μl/ml
essential amino acid solution (50 ×, Gibco BRL), 10 μl/ml nonessential amino acid solution (100 ×, Gibco BRL), 1 mM glycine, 2 mM taurine, insulin-transferrin-selenium supplement (final
concentrations of 5 μg/ml insulin, 5 μg/ml transferrin, and 5 ng/ml selenium; Sigma-Aldrich), and 6 mg/ml fatty acid-free BSA (Sigma-Aldrich). IVF oocytes were cultured in groups of 10 to 15
in 50-μl drops of mSOF at 38.5°C in an atmosphere containing 5% CO2, 5% O2, and 90% N2. Rates of early cleavage, cleavage, blastocyst formation on day 7, and
blastocyst formation on day 8 were assessed at 27 h, 72 h, 168 h, and 192 h, post-IVF. We evaluated not only the cleavage rate but also the early cleavage rate to discuss the relationship
between NT and fertility, as fast-cleaving embryos (≤ 27 h) showed higher viability and pregnancy rates than slow-cleaving embryos (> 27 h) in cattle [23 (link), 24 (link)]. Cleavage and blastocyst formation rates were calculated based on the percentages of the number of original oocytes and cleaved
oocytes, respectively.
essential amino acid solution (50 ×, Gibco BRL), 10 μl/ml nonessential amino acid solution (100 ×, Gibco BRL), 1 mM glycine, 2 mM taurine, insulin-transferrin-selenium supplement (final
concentrations of 5 μg/ml insulin, 5 μg/ml transferrin, and 5 ng/ml selenium; Sigma-Aldrich), and 6 mg/ml fatty acid-free BSA (Sigma-Aldrich). IVF oocytes were cultured in groups of 10 to 15
in 50-μl drops of mSOF at 38.5°C in an atmosphere containing 5% CO2, 5% O2, and 90% N2. Rates of early cleavage, cleavage, blastocyst formation on day 7, and
blastocyst formation on day 8 were assessed at 27 h, 72 h, 168 h, and 192 h, post-IVF. We evaluated not only the cleavage rate but also the early cleavage rate to discuss the relationship
between NT and fertility, as fast-cleaving embryos (≤ 27 h) showed higher viability and pregnancy rates than slow-cleaving embryos (> 27 h) in cattle [23 (link), 24 (link)]. Cleavage and blastocyst formation rates were calculated based on the percentages of the number of original oocytes and cleaved
oocytes, respectively.
6
Mouse Airway Progenitor Cell Co-Culture
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Mouse airway progenitor cells were co-cultured with MLg cells in Matrigel, as described previously (20 (link)). In brief, sorted airway progenitor cells were mixed with MLg cells in growth factor-reduced Matrigel (BD Biosciences) and basic medium (BM) at a 1:1 ratio. The basic medium consisted of DMEM/F12 medium (Cellgro, Manassas, VA, USA), 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.), Insulin-Transferrin-Selenium supplement (Sigma-Aldrich; Merck KGaA), 100 IU/ml penicillin and 100 µg/ml streptomycin. The cell mixture was placed in 24-well Transwell filter inserts (BD Biosciences) in a 24-well flat-bottom culture plate containing culture medium (BM + SB431542). Cultures were incubated at 37°C in a humidified atmosphere with 5% CO2, and medium was replaced every other day. Colony-forming efficiency (CFE) was determined by counting the number of colonies with a diameter of ≥100 µm in each culture and representing this number as a percentage of seeded progenitor cells.
7
Establishment of RTEC Cell Lines
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RTEC cell lines (HPT1b and FKT) and primary cultures from C57Bl/10ScN and C57Bl/6 kidneys were grown in a 50:50 mixture of DMEM/HAM’S F-12 (ATCC) supplemented with 2% FBS (Invitrogen), 5pM tri-iodothyronine (Sigma), insulin-transferrin-selenium supplement(Sigma), 3.5micrograms/mL ascorbic acid, 25ng/mL prostaglandin E1, 25ng/mL hydrocortisone, and 10ng/mL epidermal growth factor. Floxed Kidney Tubular (FKT) cell line was created after isolating RTEC from ATG7flox/flox mice. Briefly, cortex was isolated and minced with sterile blades and then filtered through a 40-micron filter before being pelleted and then resuspended in defined media. RTEC on day 5 were recognizable by their characteristic polygonal/cobblestone morphology, refractive nature, and the tendency to form domes in areas of confluence. Cells were immortalized via transduction with the lentivector VVPW/mTert that constitutively expresses the mouse telomerase [16 (link)]. A subset of FKT cells were transduced with either VPB/Cre, expressing the Cre recombinase, or the empty blasticidin-selectable VPB lentivector [16 (link)]. Following blasticidin selection, we established the FKT+ cells expressing the wild-type Atg7 and the FKT- variant cell line in which the Atg7 gene was ablated.
8
C2C12 and 293T Cell Culture Protocol
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Cell culture. Mouse myoblast c2c12 (cat. no. crl-1772) and 293T (cat. no. crl-11268) cell lines were purchased from the american Type culture collection. c2c12 and 293T cells were cultured in dMeM with high glucose supplemented with 10% FBS, 100 u/ml penicillin and 100 µg/ml streptomycin (all from Thermo Fisher Scientific, Inc.). Cultures were maintained at 37˚C at 5% CO 2 . c2c12 cells were differentiated into myotubes by replacing the growth medium with a medium containing 2% horse serum (Thermo Fisher Scientific, Inc.) with 1% penicillin-streptomycin (Thermo Fisher Scientific, inc.) and 1X insulin-transferrin-selenium supplement (Sigma-aldrich; Merck KGaa).
9
Chondrogenic Differentiation of MSCs
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MSCs were seeded at 50,000 cells/cm2 into a 12-well plate. Cells were then cultured in Chondrogenic media (10% FBS; 1% pen/strep 1% insulin-transferrin-selenium supplement (Sigma-Aldrich, Gillingham, UK), 10−7 M dexamethasone (Sigma-Aldrich, Gillingham, UK), 150 µM ascorbic-2-phosphate (Sigma-Aldrich, Gillingham, UK), 20 µM linoic acid (Sigma-Aldrich, Gillingham, UK) and 0.1 ng/mL TGF-β (Merck Millipore, Watford, UK). After 2 weeks, cells were stained with Alcian Blue (Sigma-Aldrich, Gillingham, UK) and photos were taken.
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