For air-liquid interface (ALI) cultures58 (link), 100,000 cultured epithelial cells per well were added to 0.4μm pore 24-well polyester membrane inserts (Corning) pre-coated with 0.03 mg/mL Type I bovine collagen solution (StemCell Technologies) with Pneumacult-Ex media (Stemcell Technologies, 05008) on both sides of the membrane. After 24 hours, apical media was changed to remove dead cells. After 72 hours, apical media was removed completely and basal media was changed to Pneumacult-ALI (Stemcell Technologies, 05001) supplemented with 5 mL 100x penicillin-streptomycin (Fisher), 1 mL 500x gentamicin/amphotericin B (ThermoFisher), 1 mL 0.2% heparin sodium salt in PBS (Stemcell Technologies) and 2.5 mL 200x hydrocortisone stock solution (Stemcell Technologies) and 0, 0.1, 1 or 10 ng/mL IL-13 (Biolegend). Basal media was changed every 2–3 days for 21 days, after which membranes were removed and cells dissociated with Stempro Accutase Cell Dissociateion Reagent (Gibco) for Seq-Well or flow cytometry. After following scRNA-seq data analysis pipelines described above, cell states recovered in ALI cultures (Fig. 5a ; Extended Data Fig. 9g ) were related to in vivo cell types59 (link).
Heparin sodium salt
Manufactured by STEMCELL
Heparin sodium salt is a purified product derived from animal tissues. It is an anticoagulant that inhibits the blood clotting process by inactivating several enzymes involved in the coagulation cascade.
Automatically generated - may contain errors
Lab products found in correlation
Hydrocortisone stock solution, by STEMCELL (2 mentions)
Pore 24 well polyester membrane inserts, by Corning (2 mentions)
Type 1 bovine collagen solution, by STEMCELL (2 mentions)
Pneumacult ex media, by STEMCELL (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Gentamicin amphotericin b, by Thermo Fisher Scientific (2 mentions)
Stempro accutase cell dissociateion reagent, by Thermo Fisher Scientific (2 mentions)
Il 13, by BioLegend (2 mentions)
Pneumacult ali, by STEMCELL (2 mentions)
Laminin solution, by Merck Group (1 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
Neurocult ns a proliferation kit, by STEMCELL (1 mentions)
Accutase, by Thermo Fisher Scientific (1 mentions)
Rhegf, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Mycoalert mycoplasma detection kit, by Lonza (1 mentions)
4 protocols using heparin sodium salt
1
Airway Epithelial Cell Differentiation
2
Airway Epithelial Cell Differentiation
3
Plasma-based Alzheimer's Indicator Assay
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We developed an indicator cell assay to detect AD from human plasma samples at two stages of progression: preclinical (cognitively normal patients with positive CSF AD biomarkers; N = 20) and early symptomatic (patients with mild cognitive impairment or early AD and positive CSF AD biomarkers) when compared to healthy controls (cognitively normal patients with negative CSF AD biomarkers). The study involved analysis of 18–20 samples of each class, which were processed in randomly selected batches, each containing an equal number of samples from each class. Indicator cells for this experiment were a commercially available, pan-neuronal population of glutamatergic and GABAergic neurons derived from induced pluripotent stem cells (iPSCs) (iCell Neurons from Cellular Dynamics International (CDI)). For this experiment, the neurons were thawed and plated in a 12-well dish (at 600,000 cells/well) and after culturing for 5 d, cells were exposed to 10% plasma containing 0.2 mg/mL heparin sodium salt in PBS (Stem Cell Technologies) for 24 h. RNA was isolated and used for gene expression analysis with Affymetrix human exon arrays (ST 1.0). Neurons were guaranteed free from mycoplasma contamination from CDI and were not tested again during the experiment.
4
Culturing Patient-Derived GBM Cell Lines
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Patient-derived GBM cell lines were cultured in NSA medium, composed of: reconstituted human NeuroCult™ NS-A Proliferation kit (Stem Cell Technologies) supplemented with 20µg/ml heparin sodium salt (Stem Cell Technologies), 20ng/ml rhEGF (Peprotech), 20ng/ml b-FGF (Peprotech) and 1% antibiotic/antimycotic (Life Technologies). Flasks or plates were coated with 5µg/ml laminin freshly prepared by diluting 1mg/ml laminin solution (Sigma Aldrich) in dPBS. The flasks/plates were then incubated at 37degrees for at least 1 hour. For seeding cells, laminin was aspirated and fresh medium added immediately. For maintenance, cells were scraped off and re-plated into laminin coated flasks. Accutase ( Life Technologies) was used to dissociate neurospheres and to obtain a single cell suspension, when required. All cell lines were confirmed to be mycoplasma free, based on routine testing using the MycoAlert Mycoplasma Detection Kit (Lonza).
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