Isolation of primary pneumocytes was described previously(20 ). Briefly, lungs were minced and proteolytically digested with Dispase II (Corning) and 0.01 % DNase I (Sigma-Aldrich). After negative selection of immune cells via CD16/CD32 (BD Bioscience) coated dishes, remaining pneumocytes were cultured in F12 media (Gibco) supplemented with 2 % FBS, ITS supplements (Sigma-Aldrich), 0.8 mM CaCl2, 15 mM HEPES, 0.25 (w/v) % BSA and antibiotics. Purity of cell populations was verified by immunocytochemistry using antibodies against CC-10 and SPC (Santa Cruz). 72 hours post isolation cells were transduced with rAd.Cre at a MOI of 250. For the next three days, media was replaced daily wish fresh media, and cells were harvested 5 days following adenoviral transduction in RLT lysis buffer (Qiagen). Total RNA was isolated using the RNeasy Kit (Qiagen) and analyzed using Illumina HiSeq sequencing. To test for efficient K-rasG12D recombination and Egfr deletion, we harvested cells K and KE cells two days post transduction for DNA and protein extraction and subsequent analysis.
Its supplement
Manufactured by Merck Group
Sourced in United States
ITS supplement is a laboratory reagent that provides a combination of insulin, transferrin, and selenium. It is commonly used as a supplement in cell culture media to support cell growth and viability.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Merck Group (6 mentions)
Glutamax, by Thermo Fisher Scientific (5 mentions)
Neurobasal medium, by Thermo Fisher Scientific (4 mentions)
Bovine pituitary extract, by Thermo Fisher Scientific (4 mentions)
Cholera toxin, by Merck Group (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Poly d lysine, by Merck Group (3 mentions)
D glucose, by Merck Group (3 mentions)
Insulin, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Nerve growth factor (ngf), by Thermo Fisher Scientific (2 mentions)
Matrigel, by BD (2 mentions)
Nerve growth factor (ngf), by Alomone (2 mentions)
Nih3t3 cells, by American Type Culture Collection (2 mentions)
Penicillin streptomycin, by American Type Culture Collection (2 mentions)
Purecol 5005, by Advanced BioMatrix (2 mentions)
Soy trypsin inhibitor, by Thermo Fisher Scientific (2 mentions)
Human egf, by Thermo Fisher Scientific (2 mentions)
Hek293t, by American Type Culture Collection (2 mentions)
Mda mb 231, by American Type Culture Collection (2 mentions)
32 protocols using its supplement
1
Isolation and Genetic Manipulation of Primary Pneumocytes
2
Dissociated DRG Mixed Myelinating Cultures
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Dissociated DRG mixed myelinating cultures were prepared as described (Eshed et al., 2005 (link)). Dorsal root ganglia were dissected from mouse embryos at 13.5 days of gestation, collected in Leibovitz’s L15 medium (Gibco) and dissociated with trypsin (without EDTA; Cat. # 25050, Gibco). After dissociation, 40,000 cells were plated on EtOH-washed 13 mm glass-coverslips (Thermo) pre-coated with 0.4 μg/ml Matrigel (BD Biosciences) and 10 μg/ml Poly-D-lysine (Sigma-Aldrich). DRG cultures were maintained for a day in NB medium (2% B27 (Gibco), 50 ng/ml NGF (Alomone labs) and 1% glutamax in neurobasal medium (Gibco)) and then switched to BN medium (1% ITS supplements (Sigma- Aldrich), 0.2% BSA, 4 g/L D-glucose, 50 ng/ml NGF and 1% glutamax in basal Eagle’s medium (BME; Gibco)). Myelination was induced after 10 additional days by supplementing with 50 μg/ml L-ascorbic acid (Sigma-Aldrich) and 15% heat inactivated FCS (BNC medium).
3
Primary Rat DRG Neuron Isolation and Co-Culture
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Primary rat dorsal root ganglia neurons (DRGNs) were isolated from 15-days-old Wistar rat embryo’s (Charles River), as described before (Chan et al., 2004 (link); Stancic et al., 2012 (link)), with minor modifications. Dissociated DRGNs were plated as 40 μl drops at a density of 6 × 104 cells on 13 mm coverslips (0.5 ml) that were pre-coated with PLL, followed by growth-factor-reduced matrigel (1:40 dilution; BD Bioscience). OPCs (control or transduced) were seeded onto DRGNs after 16–21 div at a 1:1 ratio in DMEM supplemented with 1% ITS supplement (Sigma-Aldrich, United States), 0.25% FBS, D+-glucose (4 mg/ml, Sigma-Aldrich, United States), L-glutamine and penicillin and streptomycin. After 2 days in co-culture, the cultures were treated with vehicle (PBS) or 50 μg/ml gpTG2. Co-cultures were maintained for up to 14 days with medium changes at every third day. GpTG2 was added upon medium changes.
4
Mouse Hepatocyte Lipid Accumulation Assay
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The mouse hepatocyte AML12 cell line (ATCC; Manassas, VA, USA) was cultured in high-glucose Dulbecco’s modified Eagle medium (DMEM; HyClone, Logan, UT, USA) with 10% fetal bovine serum (FBS; HyClone), 1% penicillin/streptomycin (Invitrogen; Carlsbad, CA, USA), ITS supplement (Sigma), and 40 ng/mL dexamethasone. The mouse macrophage RAW264.7 cell line was cultured in DMEM medium with 10% FBS and 1% penicillin/streptomycin. All cells were grown in an incubator maintained at 37 °C with 5% CO2. Recombinant irisin was purchased from Phoenix Pharmaceuticals (Burlingame, CA, USA) and recombinant human MD2 (rhMD2) was purchased from R&D Systems (Minneapolis, MN, USA). Lipid accumulation in the hepatocytes was observed by Oil Red O staining as described previously [28 (link)].
5
Cell Culture Protocols for Cancer Research
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HEK293T (ATCC CRL-3216), A549 (CCL-185), MDA-MB-231 (ATCC HTB-26), and KPT1 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 100 IU/mL of penicillin/streptomycin. KP cells4 were a kind gift from Dr. Tyler Jacks (MIT). PC9 cells were a kind gift from Dr. Harold Varmus (Weill Cornell) and cultured in RPMI supplemented with 10% fetal bovine serum (FBS) and 100 IU/mL of penicillin/streptomycin. NIH3T3 cells (ATCC CRL-1658) were cultured in DMEM supplemented with 10% fetal calf serum (FCS) and 100 IU/mL of penicillin/streptomycin. Pancreatic ductal epithelial cells (PDECs)5 were a kind gift from Dr. Dafna Bar-Sagi (New York University) and cultured in collagen-coated plates (100 μg/mL PureCol 5005, Advanced Biomatrix) with Advanced DMEM/F12 supplemented with 10% FBS (Gibco), 100 IU/mL of penicillin/streptomycin (Gibco), 100 mM Glutamax (Gibco), ITS Supplement (Sigma), 0.1 mg/mL soy trypsin-inhibitor (Gibco), Bovine Pituitary Extract (Gibco), 5 nM T3 (Sigma), 100 μg/mL Cholera toxin (Sigma), 4 μg/mL Dexamethasone (Sigma), and 10 ng/mL human EGF (Preprotech).
6
Isolation and Culture of Ovarian Follicles
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Frozen-thawed ovaries were moved to 35 mm dish (SUMILON, Tokyo, Japan) containing 3 ml of DMEM/F12 medium. Secondary follicles were isolated from frozen-thawed ovaries by the puncture with 26G 1/2 needle (Terumo corporation) under the stereo microscope. The secondary follicles were selected by glass pipette. After washing with PBS (-), the secondary follicles were placed in 0.5% (w/v) sodium alginate (Wako)/PBS (Ca2+-and- Mg2+ -free) and then placed in 50 mM CaCl2/saline to cross-link the alginate. Ten of secondary follicles were placed in one alginate bead and were cultured for 6 days in the medium (DMEM/F12 containing penicillin and streptomycin) containing 10 ng/ml ovine FSH (NIDDK, Torrance, CA, USA) and ITS Supplement (Sigma) in the presence of 1% (v/v) FBS. The culture medium was changed every 2 days and these secondary follicles were captured using a Keyence BZ-9000 microscope (Keyence Co., Osaka, Japan). The area of each secondary follicle was measured by a BZ analyzer.
7
Engineered Skin Substitutes for RDEB
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ESS were prepared essentially as previously described21 (link). Briefly, fibroblasts (0.5 × 106/cm2) were inoculated onto
sterile collagen-glycosaminoglycan scaffolds supported at the air–liquid interface using
polyvinyl acetal sponges. Keratinocytes (1.0 × 106/cm2) were
inoculated onto the dermal substrates 24 h later and ESS were transferred to cotton pads
supported by perforated stainless steel platforms for incubation at the air–liquid
interface for 2 weeks (37°C, 5% CO2). Culture medium for ESS consisted of
DMEM/F12 (Sigma-Aldrich) supplemented with 1 mM strontium chloride, 0.3% FBS, 1× ITS
Supplement (Sigma-Aldrich), 10 µg/ml linoleic acid, 0.1 mM AA2P, 20 pM triiodothyronine,
0.5 µg/ml hydrocortisone, 5 ng/ml keratinocyte growth factor (Peprotech), 1 ng/ml basic
fibroblast growth factor (Peprotech), and 1× PSF19 (link). Four groups of grafts were prepared: group 1, RDEB fibroblasts and RDEB
keratinocytes; group 2, RDEB fibroblasts and normal keratinocytes; group 3, normal
fibroblasts and RDEB keratinocytes; and group 4, normal fibroblasts and normal
keratinocytes.
sterile collagen-glycosaminoglycan scaffolds supported at the air–liquid interface using
polyvinyl acetal sponges. Keratinocytes (1.0 × 106/cm2) were
inoculated onto the dermal substrates 24 h later and ESS were transferred to cotton pads
supported by perforated stainless steel platforms for incubation at the air–liquid
interface for 2 weeks (37°C, 5% CO2). Culture medium for ESS consisted of
DMEM/F12 (Sigma-Aldrich) supplemented with 1 mM strontium chloride, 0.3% FBS, 1× ITS
Supplement (Sigma-Aldrich), 10 µg/ml linoleic acid, 0.1 mM AA2P, 20 pM triiodothyronine,
0.5 µg/ml hydrocortisone, 5 ng/ml keratinocyte growth factor (Peprotech), 1 ng/ml basic
fibroblast growth factor (Peprotech), and 1× PSF19 (link). Four groups of grafts were prepared: group 1, RDEB fibroblasts and RDEB
keratinocytes; group 2, RDEB fibroblasts and normal keratinocytes; group 3, normal
fibroblasts and RDEB keratinocytes; and group 4, normal fibroblasts and normal
keratinocytes.
8
Culturing Cortical Neurons from Newborn Mice
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Cortical neurons from newborn C57BL/6NCrl mice of both sexes were prepared and maintained as described previously [15 (link)]. In brief, 40,000 cells per well were seeded into Poly-D-Lysine coated 96-well plates (BD Biocoat 356,640) and were grown in Neurobasal-A medium (Life Technologies) supplemented with B27 (Life Technologies), 0.5 mM glutamine, and 1% rat serum. To prevent the proliferation of glial cells, cytosine β-D-arabinofuranoside (Sigma-Aldrich, 2.8 μM) was added on day in vitro (DIV) 3. On DIV 8 growth medium was exchanged to a defined minimal medium consisting of a mixture of buffered salt-glucose-glycine (SGG) solution [10 mM Hepes (pH 7.4), 114 mM NaCl, 26.1 mM NaHCO3, 5.3 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 30 mM glucose, 1 mM glycine, 0.5 mM sodium pyruvate, and 0.001% phenol red] and phosphate-free Eagle’s minimum essential medium (MEM, Life Technologies) (9:1 vol:vol), supplemented with insulin (7.5 μg/ml), transferrin (7.5 μg/mI), and sodium selenite (7.5 ng/ml) (ITS supplement, Sigma-Aldrich). Experiments were performed after a culturing period of 10–12 DIV.
9
Engineered Skin Substitutes: Fibroblast and Keratinocyte Coculture
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ESS were prepared essentially as previously described [39 (link)] with the following modifications. Fibroblasts were inoculated at 0.5 X 106/cm2 onto sterile collagen-glycosaminoglycan scaffolds supported at the air-liquid interface using polyvinyl acetal sponges. Keratinocytes were inoculated onto the dermal substrates 24 hours later at 1.0 X 106/cm2 and ESS were transferred to cotton pads supported by perforated stainless steel platforms for incubation at the air-liquid interface (37°C, 5% CO2). Culture medium for ESS consisted of DMEM/F12 (Sigma-Aldrich) supplemented with 1 mM strontium chloride, 0.3% FBS, 1X ITS Supplement (Sigma Aldrich), 10 μg/ml linoleic acid, 0.1 mM AA2P, 20 pM triiodothyronine, 0.5 μg/ml hydrocortisone, 5 ng/ml keratinocyte growth factor (Peprotech, Rocky Hill NJ), 1 ng/ml basic fibroblast growth factor (Peprotech), and 1X PSF [37 (link)]. ESS were cultured in vitro for 10 days prior to transplantation to mice.
10
Podocyte Migration Assay with CLCN5 Knockdown
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A migration assay was done as described previously with minor modification.18 (link), 19 Control and CLCN5 knockdown podocytes were grown in 35-mm glass-bottom culture dishes (MatTek Corporation) until a confluent cell monolayer was achieved. The cells were serum-starved in RPMI 1640 medium for 8 to 12 hours. A scratch wound was created using a 1- to 10-μl pipette tip. Wounds were created with 2 strokes at a 90-degree angle and washed twice with phosphate-buffered saline to remove all the suspended cells in the medium. The cells were then cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, ITS supplement (Sigma-Aldrich), and 200 U/ml penicillin and streptomycin (Roche Applied Science) at 33°C for 12 hours. Images were taken at different time points (0, 6, and 10 hours). The experiment was performed 3 times, and the rate of migration was calculated using ImageJ software.
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