Cells were purchased from ATCC (Manassas, VA). Immortalized, esophageal, squamous epithelial cells, EPC2 were a generous gift from Dr. Anil Rustgi, University of Pennsylvania. Mouse tissue was a generous gift from Dr. Landon Inge, St. Joseph’s Hospital and Medical Center. Cells were maintained at 37 °C in a humidified incubator with 5% CO2 according to ATCC protocols. The cell lines used were: CP-A (CRL-4027), EPC2, HeLa (CCL-2.2), Swiss 3T3 (CCL-92), MDA-MB-231 (HTB-26), HME1 (CRL-4010), MCF 10A (CRL-10317), MCF7 (HTB-22). EPC2 cells were maintained according to the ATCC protocol for CP-A cells. Primary cells were prepared from lung tissue from a K-ras G12D mouse model of lung cancer. To prepare primary cell cultures, fresh tissue samples were washed 3 times with RPMI containing 5 ng/mL amphotericin B, then minced into 1 to 2 mm pieces. Samples were dissociated with collagenase III in Chang medium (Irvine Scientific, Santa Ana, CA, C101) supplemented with Chang medium supplement (Irvine Scientific, C108) at 37 °C for 3 hours with occasional shaking. Cells were pelleted and expended in Chang medium, changed every 3 days. Epithelial and fibroblast cell populations were isolated by multiple rounds of differential trypsinization.
Chang medium
Manufactured by Irvine Scientific
Sourced in United States
Chang medium is a cell culture medium formulated for the growth and maintenance of human Chang conjunctival cells. It provides the necessary nutrients and growth factors to support the in vitro culture of these cell types. The composition and properties of Chang medium are designed to meet the specific requirements for cultivating Chang conjunctival cells.
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Lab products found in correlation
α mem medium, by Thermo Fisher Scientific (1 mentions)
Basic fibroblast growth factor (bfgf), by R&D Systems (1 mentions)
Anti human cd73, by BD (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Biotin 16 dutp, by Roche (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Memα medium, by Thermo Fisher Scientific (1 mentions)
Bio amf 2, by Sartorius (1 mentions)
6 protocols using chang medium
1
Primary Culture of Mouse Lung Cells
2
Antibody Labeling and Cell Culture Reagents
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FITC or phycoerythrin (PE)-conjugated monoclonal antibodies anti-human CD73 and CD90 were from BD Biosciences (Franklin Lakes, NJ, U.S.A.). Monoclonal antibodies anti-mouse CD34 were from Biolegend (San Diego, CA, U.S.A.). Polyclonal antibody anti-human Lgr6 was from Novus Biologicals (Littleton, CO, U.S.A.). Polyclonal antibodies anti-human KGF and bFGF) were from R&D Systems (Minneapolis, MN, U.S.A.). Atelocollagen membranes (CM) were purchased from Cosmo Bio (Tokyo, Japan). α-MEM medium was from Life Technologies (U.S.A.) and Chang medium was from Irvine Scientific (U.S.A.).
3
3D GelMA Scaffold for HL-MSC Co-culture
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GelMA scaffolds were dipped in MSC medium, i.e., 1 mL MEM-α containing desossiribonucleotides supplemented with 2 mM glutamine, 1% penicillin/streptomycin (GIBCO, Thermo Scientific, Rochester, NY, USA) and 1% Chang medium (Irvine Scientific, Wicklow, Ireland), 1 h before cell seeding [30 (link),33 (link)]. A mixture of 3 × 105 L428 HL cells (DSMZ GmbH, Braunschweig, Germany) in 100 µL of RPMI1640 medium and 2 × 105 HL-derived LN-MSC, obtained as described [33 (link)], in 100 µL of MSC medium were seeded onto the pre-wet GelMA scaffolds in a 24 w plate and kept 2 h at 37 °C in a 5% CO2 humidified incubator, then 1 mL of MSC/RPMI1640 (50% v/v) medium was added and samples cultured up to 14 d. To avoid cell starvation, half medium was replaced every two days. At 7 d and 14 d, repopulated GelMA scaffolds were fixed in 4% buffered formalin or Histochoice and embedded in paraffine for IHC. Parallel samples underwent Young’s modulus measurement and SEM analysis.
4
Preparation of R-banded Chromosomes and FISH
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The preparation of R-banded chromosomes and FISH was performed as previously described [38] (link), [39] (link). Fibroblasts obtained from tails of male T. muenninki and T. osimensis were cultured in Chang Medium (Irvine Scientific) and/or DMEM supplemented with 12% fetal bovine serum at 37°C in an atmosphere of 5% CO2. BAC clones of T. muenninki and T. osimensis containing SOX9 and TESCO were used as probes. The BAC clones were labeled by nick translation with biotin-16-dUTP (Roche Diagnostics) according to the manufacturer's protocol. A mixture (20 µl) containing the labeled DNA was placed on the denatured chromosome slides and covered with parafilm, and the slides were incubated overnight at 37°C. After being washed in 4× SSC, the slides were incubated under parafilm for 1 h with fluoresceinated avidin (Roche) at 1∶500 dilution in 1% BSA/4× SSC. The slides were washed on a shaker with 4× SSC, 0.01% Nonidet P-40y in 4× SSC, and 4× SSC, each for 10 min. The chromosome slides were stained with propidium iodide (0.75 ug/ml).
5
Isolation and Characterization of Amniotic Fluid Stem Cells
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The study was conducted in accordance with the guidelines on good clinical practice and with the ethical standards for human experimentation established by the 1964 Declaration of Helsinki. All subjects gave informed consent before being included in the study. The study protocol was reviewed and approved by Institutional Review Board of Siriraj Hospital, Mahidol University, Bangkok, Thailand [Protocol No. 103/2553(EC3)]. Amniotic fluid was collected from the Department of Obstetrics and Gynecology, Faculty of Medicine Siriraj Hospital. Briefly, amniotic fluid samples were obtained by amniocentesis performed for routine fetal genetic diagnosis at 16–20 weeks of gestation, centrifuged at 2100RPM for 5 min, and the pellet retrieved for cell line establishment. AFSC were cultured in MEMα medium (Gibco, Invitrogen, Carlsbad, MA, USA) supplemented with 15% embryonic stem cell qualified fetal bovine serum (ES-FBS), 1% L-glutamine, 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA), 20% Chang medium (Irvine Scientific, Santa Ana, CA, USA) at 37 °C with stable 5% CO2 in normoxic conditions28 (link). Cell quantity, morphology and expression of Oct-4, CD29, CD34, CD44, CD45, CD73, CD90 and CD105 were evaluated. Chromosomal stability was assessed by karyotype (Supplementary data – Methods ).
6
Amniocyte Culture and DNA Extraction
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The amniocytes were centrifuged and half of the pellet suspended in CHANG medium (Irvine Scientific, Santa Ana, CA, United States) while the other half was suspended in BioAMF-2 (Biological Industries, Beit-HaEmek, Israel) and cultured in 25 cm flasks for 10–14 days. When cells reached confluence, they were either harvested for DNA extraction or trypsinized for the next passage. Cells harvested at the fifth and at seventh; by the eighth passage, the culture failed.
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