DRG neurons were prepared as previously reported (Hotta et al., 2019a (link); Hori et al., 2022 (link)). Rats were euthanized by bilateral thoracotomy and removal of the heart under deep isoflurane sedation (4–5% in 100% oxygen). The adequacy of anesthesia was verified by lack of a withdrawal response to tail pinch. Collected DRGs from rats were digested with collagenase IV (1.0 mg/mL, Sigma) for 30 min and trypsin-EDTA (0.05%, Sigma) for 5 min each at 37˚C. Following, the enzyme reaction was terminated by using trypsin inhibitor (0.08 mg/mL, Thermo Fisher Scientific) at RT. DRGs were then washed with Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12 (Thermo Fisher Scientific) supplemented with nerve growth factor (0.1 µg/mL, NGF-7S, Sigma), fetal bovine serum (5%, Sigma), Glutamax (1%, Thermo Fisher Scientific), glucose (0.8%, Sigma), penicillin-streptomycin (10 µL/mL, Sigma), and Dulbecco’s phosphate buffered saline (Sigma). DRGs were placed on glass coverslips coated with poly-L-lysine (0.1 mg/mL, Sigma) and laminin (0.13%, Thermo Fisher Scientific) after being suspended and dissociated in the supplemented DMEM/Ham’s F-12 solution using a fire-polished Pasteur pipette. DRGs were maintained at 37˚C in a CO2 incubator replacing with fresh supplemented medium at least every 2 days up to the day of current recording.
Dmem ham s f12
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, United Kingdom, Japan, Switzerland, France, Sweden, Australia
DMEM/Ham's F12 is a cell culture medium that provides a balanced environment for the growth and maintenance of a wide variety of cell types, including adherent and suspension cell lines. The medium is a combination of Dulbecco's Modified Eagle's Medium (DMEM) and Ham's F12 nutrient mixture, which together provide a comprehensive set of amino acids, vitamins, salts, and other essential components required for cell proliferation and survival.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (41 mentions)
Hydrocortisone, by Merck Group (19 mentions)
Penicillin, by Thermo Fisher Scientific (16 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (16 mentions)
Rpmi 1640, by Thermo Fisher Scientific (15 mentions)
Streptomycin, by Thermo Fisher Scientific (15 mentions)
Insulin, by Merck Group (14 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (14 mentions)
Penicillin streptomycin, by Merck Group (12 mentions)
Penicillin, by Merck Group (10 mentions)
Streptomycin, by Merck Group (9 mentions)
L glutamine, by Thermo Fisher Scientific (9 mentions)
Glutamax, by Thermo Fisher Scientific (7 mentions)
Hepes, by Thermo Fisher Scientific (7 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (7 mentions)
Fetal bovine serum (fbs), by Merck Group (6 mentions)
Amphotericin b, by Merck Group (6 mentions)
L glutamine, by Merck Group (6 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (6 mentions)
Cholera toxin, by Merck Group (6 mentions)
213 protocols using dmem ham s f12
1
Preparation of Rat DRG Neurons
2
Culturing Isolated UCB Mononuclear Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolated UCB mononuclear cells were cultured in RPMI 1640 (Hyclone Laboratories, Logan, UT, USA), a mixture of DMEM/Ham’s F-12 (Gibco, Grand Island, NY, USA; 2:1 v/v), and AIM V serum-free (Gibco) basal medium. RPMI 1640 medium was supplemented with 10% heat-inactivated FBS (Gibco), 100 U/mL penicillin, 100 μg/mL streptomycin (Lonza, Walkersville, MD, USA), and 4 mmol/L glutamine (Gibco) (RC). DMEM/Ham’s F-12 (2:1) was supplemented with 10% heat-inactivated human AB serum (Thermo Fisher Scientific, Waltham, MA, USA), 25 µM beta-mercaptoethanol (Thermo Fisher Scientific), 50 µmol/L ethanolamine (Sigma-Aldrich, St. Louis, MO, USA), 20 µg/L ascorbic acid (Sigma-Aldrich), and 5 ng/L sodium selenite (Sigma-Aldrich) (DS). AIM V was supplemented with 5% ICSR (Life Technologies, Grand Island, NY, USA).
3
Murine Lung Slice Culture for Fibrosis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PCLSs were generated as previously described [50 (link)]. Briefly, warm, low gelling temperature agarose (2% by weight, Sigma) in sterile DMEM/Ham’s F12 (Gibco) was infiltrated into mouse lung through trachea. After the trachea being ligated with thread to retain the agarose inside the lung, the whole lung was excised and cooled on ice for 10 min to allow gelling of the agarose. Then the separated lobes were cut with a vibratome (VT1000 S, Leica, Buffalo Grove, IL, USA) to a thickness of 300 μm. The PCLSs were cultivated in DMEM/Ham’s F12 (Gibco) with 0.1% FBS (Gibco), 100 μg/ml streptomycin, 100 U/ml penicillin, 0.25 μg/ml amphotericin B, and 10 mmol/l HEPES (Sigma Aldrich) at 37°C in a humidified 5% CO2 atmosphere. For control cocktail (CC) or fibrosis cocktail (FC) treatment, 50 ng/ml rKL was added to serum-free medium 12 h after serum withdrawal. After 24 h of klotho pre-incubation, mouse PCLSs were incubated with CC or FC for another 48 h. Both the CC and FC were prepared as previously described [21 (link)] (28314802). In brief, FC contains 5 ng/ml TGF-β (PeproTech), 5 μM PDGF-AB (PeproTech), 10 ng/ml TNF-α (PeproTech), and 5 μM LPA (Cayman Chemical, Ann Arbor, MI, USA).
4
Optic Cup Structures from iPSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Three-dimensional optic cup structures protocols were adapted from previous protocols11 (link), 12 (link). Confluent iPSC colonies were dissociated using EDTA (0.5 M, Invitrogen), resuspended in Essential6 media (Gibco) and allowed to form embryoid bodies in non-adherent 10 cm petri dishes (Sterilin) at 37 °C on an orbital shaker (Stuart). Twenty-four hours later they began patterning for 12 days with reducing knockout serum replacement (KOSR; 20% for 5 days, 15% until day 9, 10% until day 37), 1:50 B27 (PAA), 5 ng/ml rhIGF-1 (Sigma), 3 µm IWR1e and 1% Geltrek in DMEM/Ham’s F-12 (Gibco). From day 12–18, cultures were incubated with reducing KOSR, 1:50 B27 (PAA), 5 ng/ml rhIGF-1 (Sigma), 10% FCS, 100 nm SAg and 1% Geltrek in DMEM/Ham’s F-12 (Gibco). From day 18–37, DMEM: F-12 was supplemented with 10% KOSR, 1:50 B27 (PAA), 5 ng/ml rhIGF-1 (Sigma) and 1% Geltrek. From day 37 onwards, DMEM: F-12 was supplemented with 1:50 B27 (PAA), 1:100 N2 (Gibco), 10 ng/ml rhIGF-1 (Sigma) and 1% Geltrek.
5
Isolation and Culture of Adipose-Derived MSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolation and culture of human, adipose-derived MSCs With the approval of the local ethics committee, lipoaspirates were obtained from 3 different donors, 2 female and 1 male, age 20-45 years. Samples (~ 200 mL) were mixed with an equal volume of saline and digested at 37 °C for 1 h with 0.075 % (w/v) collagenase (Sigma-Aldrich) in PBS. Digestion was stopped by the addition of an equal volume of DMEM/Ham's F12 (Gibco) containing 10 % (v/v) FBS (Gibco) and the digested material centrifuged. Supernatants were discarded and cells re-suspended in erythrocyte lysis buffer, after which the nucleated cells were resuspended in DMEM/Ham's F12 (Gibco) containing 10 % (v/v) FBS (Gibco), 100 µg/ mL primocin (InvivoGen, San Diego, CA, USA) and 1 ng/mL basic fibroblast growth factor (Sigma-Aldrich). Medium was changed every other day and cells sub-cultured when confluent. Cell growth was measured by MTS assay. Cells at passages 2-4 were used for experiments.
6
Isolation and Immortalization of Mouse Lung Microvascular Endothelial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary mouse lung microvascular endothelial cells (mLMEC) were isolated from WT C57/BL6 adult mice. mLMECs were twice subject to magnetic-activated cell sorting to positively select for endomucin+ ECs as previously described by Reynolds and Hodivala-Dilke (37 (link)). ECs were immortalized using polyoma-middle-T (PyMT) antigen by retroviral transfection as previously described by Robinson and colleagues (38 (link)). Immortalized mLMECs were cultured in media composed of a 1:1 mix of low-glucose Ham's F-12:DMEM (Thermo Fisher Scientific) medium supplemented with 10% FBS, 100 units/mL penicillin/streptomycin, 2 mmol/L glutamax, 50 μg/mL heparin (H3393, Sigma). ECs were grown at 37°C in a humidified incubator with 5% CO2 unless otherwise stated. For experimental analyses, plasticware was coated using 10 μg/mL human plasma fibronectin (FN; Millipore). Passaging of ECs did not exceed 18.
EC stimulation was achieved using 30 ng/mL VEGF-A164 (VEGF-A; mouse equivalent of VEGF-A165) after 3 hours incubation in serum-free medium (OptiMEM; Thermo Fisher Scientific). VEGF-A was made in-house as previously described by Krilleke and colleagues (39 (link)).
EC stimulation was achieved using 30 ng/mL VEGF-A164 (VEGF-A; mouse equivalent of VEGF-A165) after 3 hours incubation in serum-free medium (OptiMEM; Thermo Fisher Scientific). VEGF-A was made in-house as previously described by Krilleke and colleagues (39 (link)).
7
Primary Culture of Mouse lDRG Neurons
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary cultures of mouse lDRG neurons were done as described before for DRG cultures [76] with the following modifications: For each lDRG culture, 4 Complete Freund's Adjuvant (CFA)-or vehicle-injected wild type mice were sacrificed by CO 2 -inhalation and only ipsilateral lumbar DRG 1-5 were dissected. The growth medium Hams' F12/DMEM (ThermoFisher Scientific) was supplemented with 10% horse serum (ThermoFisher Scientific) and 100 ng/mL NGF (R&D Systems, Minneapolis, MN). Neurons were plated on PDL (1 mg/mL) and Laminin (20 µg/mL)-coated coverslips.
Transient transfection of lDRG neurons with 500 nM AllStar Negative Control siRNA (#SI03650318, Qiagen, Hilden, Germany) or Vti1b siRNA (FlexiTube GeneSolution #GS53612, Qiagen) was done via electroporation using the 4D-Nucleofector™ System (X unit, Lonza AG, Basel, Switzerland) with P3 Primary neuron nucleofector solution with supplement (Lonza AG), program DC104. Afterwards RPMI medium (ThermoFisher Scientific) with low calcium was added for recovery and cells were plated on PDL/Laminin-coated coverslips. After a 15 min recovery period at 37°C, 1 mL of DMEM/Hams' F12 + growth factors was added per well. To reduce toxicity, half of the media was refreshed after 2 h at 37°C, 5% CO 2 . Cells were used 72 h after siRNA transfection in various in vitro assays (see below).
Transient transfection of lDRG neurons with 500 nM AllStar Negative Control siRNA (#SI03650318, Qiagen, Hilden, Germany) or Vti1b siRNA (FlexiTube GeneSolution #GS53612, Qiagen) was done via electroporation using the 4D-Nucleofector™ System (X unit, Lonza AG, Basel, Switzerland) with P3 Primary neuron nucleofector solution with supplement (Lonza AG), program DC104. Afterwards RPMI medium (ThermoFisher Scientific) with low calcium was added for recovery and cells were plated on PDL/Laminin-coated coverslips. After a 15 min recovery period at 37°C, 1 mL of DMEM/Hams' F12 + growth factors was added per well. To reduce toxicity, half of the media was refreshed after 2 h at 37°C, 5% CO 2 . Cells were used 72 h after siRNA transfection in various in vitro assays (see below).
8
Isolation of Placenta-Derived Mesenchymal Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The utilized human normal term placenta (38–40 weeks) had no evidence of any medical, obstetrical, or surgical complication. All participants provided written informed consent prior to placenta collection. The collection and use of placenta were approved by the Institutional Review Board of the Kangnam CHA General Hospital, Seoul, Korea (07–18). PD-MSCs were isolated as described previously11 (link). Briefly, the fetal membrane was removed from the chorionic plate of each placenta, and cells were treated with 0.5% collagenase IV (Sigma-Aldrich) for 30 min at 37 °C. The harvested cells were plated (2 × 105 cells/cm2) with Ham’s F-12/DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) (Life Technologies).
9
Isolation and Characterization of Pancreatic Stellate Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Pancreatic stellate cells (hPSC5) were isolated from the resected pancreatic tissue of a patient undergoing operation for pancreatic cancer as previously reported, 23 under the approval of the Ethics Committee of Tohoku University Graduate School of Medicine.
In brief, the resected human pancreatic tissue was minced with scissors and shaken in a solution containing 0.03% collagenase P (Roche, Basel, Switzerland), 0.02% pronase (Roche), and 0.1% DNase I (Roche) at 37°C for 30 min. Digested tissue was pipetted through the narrow orifices and centrifuged. After washing, cells were seeded on tissue culture dishes coated with type I collagen (Corning) and maintained in Ham's F12/DMEM (Life Technologies) supplemented with 10% FBS, 100 U/mL of penicillin, and 100 g/mL of streptomycin. hPSC5 cells were cultured in 6-well and 96-well plates (3.0 10 4 and 1.0 10 3 cells, respectively) and stimulated with 1 ng/mL rat recombinant TNF-α (R&D Systems, Minneapolis, MN, USA) in AMSC-conditioned medium or serum-free α-MEM. Following a 3-h incubation, total RNA was collected and expression levels of human MCP-1, IL-8, TGF-, α-SMA were measured by qRT-PCR. After 24 h of incubation, cell proliferation was tested using CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega).
In brief, the resected human pancreatic tissue was minced with scissors and shaken in a solution containing 0.03% collagenase P (Roche, Basel, Switzerland), 0.02% pronase (Roche), and 0.1% DNase I (Roche) at 37°C for 30 min. Digested tissue was pipetted through the narrow orifices and centrifuged. After washing, cells were seeded on tissue culture dishes coated with type I collagen (Corning) and maintained in Ham's F12/DMEM (Life Technologies) supplemented with 10% FBS, 100 U/mL of penicillin, and 100 g/mL of streptomycin. hPSC5 cells were cultured in 6-well and 96-well plates (3.0 10 4 and 1.0 10 3 cells, respectively) and stimulated with 1 ng/mL rat recombinant TNF-α (R&D Systems, Minneapolis, MN, USA) in AMSC-conditioned medium or serum-free α-MEM. Following a 3-h incubation, total RNA was collected and expression levels of human MCP-1, IL-8, TGF-, α-SMA were measured by qRT-PCR. After 24 h of incubation, cell proliferation was tested using CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega).
10
Culturing FAO Cells in DMEM/F-12
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
FAO cells were cultured in DMEM/F-12 Ham's (Gibco BRL/Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Biowest, Nuaillé, France), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco BRL/Invitrogen) at 37 °C in 5% CO2.
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!