Spheroid cultures were grown as described, with minor changes 39 (link). Briefly, 1 × 104 cells/mL were counted and resuspended carefully using a 25G syringe needle to obtain a single-cell suspension. The cells were pelleted, washed with cold PBS, and syringe-filtered again to ensure that it remains as a single-cell suspension. Cells were then plated onto Ultra-Low attachment 6-well plates (Corning, NY, USA) with 2 mL DMEM/F12 media (WelGENE Inc.) supplemented with 1% PSA, 2% B27, 10 ng/mL FGFb, 20 ng/mL EGF, 5 μg/mL insulin, and 4 μg/mL heparin. Cells were maintained at 37 °C in a 5% CO2 incubator. The number of spheroids with a diameter greater than 50 μm was regularly counted, and when there were approximately 100 spheroids of this diameter, they were collected by gentle centrifugation, dissociated, and then passaged for the assessment of self-renewal.
Dmem f12 media
Manufactured by Welgene
Sourced in United States
DMEM/F12 is a cell culture medium formulation commonly used for the growth and maintenance of a variety of mammalian cell lines. It provides a balanced salt solution, amino acids, vitamins, and other nutrients required for cell proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Ultra low attachment 6 well plate, by Corning (3 mentions)
L glutamine, by Welgene (2 mentions)
Insulin transferrin selenium solution, by Thermo Fisher Scientific (2 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
Hepes, by Welgene (1 mentions)
Mle 12 cells, by American Type Culture Collection (1 mentions)
Streptomycin sulfate, by Welgene (1 mentions)
Amphotericin b, by Welgene (1 mentions)
Wi38 human lung fibroblasts, by American Type Culture Collection (1 mentions)
Fetal bovine serum (fbs), by Welgene (1 mentions)
Beas 2b cell, by American Type Culture Collection (1 mentions)
Penicillin, by Welgene (1 mentions)
Basic fibroblast growth factor (bfgf), by BD (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Accumax, by Merck Group (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Poly l ornithine, by Merck Group (1 mentions)
24 well culture plate, by Merck Group (1 mentions)
Flex camera, by Nikon (1 mentions)
Growth factor reduced matrigel, by BD (1 mentions)
7 protocols using dmem f12 media
1
Sphere Culture Protocol for Stem Cells
2
Paclitaxel Impacts Spheroid Formation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Considering the different growth rates of two cell lines, 8 × 104 A2780 cells or and 2.5 × 105 R182 cells were seeded onto the Ultra-low attachment 6 well plate (Corning Incorporated Life Science, Lowell, MA, USA) in serum-free DMEM/F12 media (Welgene) supplemented with 20 ng/mL human recombinant epidermal growth factor (EGF; BD Biosciences), 20 ng/mL basic fibroblast growth factor (bFGF; Peprotech, Rocky Hill, NJ, USA), and 2% (v/v) B27 (Thermo Fisher scientific, Waltham, MA, USA). Cells were then treated with 1 μM paclitaxel for 4 days, and then spheroid formation of cells were measured under the microscope (Motic Electric Group Co Ltd, Xiamen, China).
3
Cell Culture Protocols for RPE, Neurons, Macrophages, and Alveolar Epithelial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human retinal pigment epithelial (RPE) cells were kindly provided by Dr. Jeong Hun Kim (College of Medicine, Seoul National University, Seoul, Korea). The murine hippocampal neuronal cell line HT-22 was kindly provided by Dr. Dong Gyu Jo (College of Pharmacy, Sungkyunkwan University, Suwon, Korea). The murine macrophage cell line RAW 264.7 was kindly provided by Dr. Sang Kook Lee (Seoul National University). Murine alveolar epithelial MLE-12 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). RPE, HT-22, and RAW 264.7 cells were cultured in DMEM supplemented with 10% FBS and antibiotics (all from Welgene, Inc., Gyeongssan, Korea). MLE12 cells were cultured in HITES medium [DMEM-F12 media (Welgene) containing 1x insulin-transferrin-selenium solution (Thermo Fisher Scientific), 10 nM hydrocortisone, 10 nM β-estradiol, 10 mM HEPES, and 2 mM L-glutamine (Welgene)] supplemented with 2% FBS and antibiotics. Cells were incubated at 37°C with 5% CO2 in a humidified atmosphere.
4
Spheroid Culture Methodology for Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Spheroid cultures were performed according to the previous procedure with minor changes [30 (link)]. Briefly, 1 × 104 cells/mL were counted and resuspended carefully using a 25 G syringe needle to obtain a single-cell suspension. The cells were pelleted, washed with cold PBS, and syringe-filtered again to ensure a single-cell suspension. Cells were plated onto Ultra-Low attachment 6-well plates (Corning, NY, USA) with 2 mL DMEM/F12 media (WelGENE Inc., Daegu, Korea) supplemented with 1% PSA, 2% B27, 10 ng/mL FGFb, 20 ng/mL EGF, 5 μg/mL insulin, and 4 μg/mL heparin. Cells were maintained at 37 °C in a 5% CO2 incubator on designated days. The number of spheroids with a diameter greater than 50 μm was regularly counted.
5
Culturing Diverse Lung Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BEAS-2B cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Human bronchial epithelial (HBEL)/p53i cells were kindly provided by Dr. John Minna (University of Texas Southwestern Medical Center, Dallas, TX, USA)13 (link), and 1799, 1198, and 1170-I cells were kindly provided by Dr. Curtis C. Harris (National Cancer Institute, USA)14 (link). These cells were cultured in K-SFM (Thermo Fisher Scientific, Waltham, MA, USA) with 5 ng/mL recombinant epidermal growth factor (EGF) and 50 μg/mL bovine pituitary extracts. MLE-12 murine alveolar epithelial cells, THP-1 human monocytes/macrophages, and Wi38 human lung fibroblasts were purchased from ATCC. MLE-12 cells were cultured in HITES medium [DMEM-F12 media (Welgene, Inc., Gyeongssan-si, Republic of Korea) containing 1× insulin-transferrin-selenium solution (Thermo Fisher Scientific), 10 nM hydrocortisone, 10 nM β-estradiol, 10 mM HEPES, and 2 mM L-glutamine (Welgene)] with 2% fetal bovine serum (FBS) (Welgene) and 1× antibiotic-antimycotic solution (100 units/mL penicillin, 10 mg/mL streptomycin sulfate, and 25 μg/mL amphotericin B in 0.85% NaCl solution; Welgene). THP-1 and Wi38 cells were cultured in RPMI 1640 medium and DMEM, respectively, with 10% FBS and 1× antibiotic-antimycotic solution. Cells were maintained at 37 °C in a humidified atmosphere with 5% CO2.
6
Neurosphere Culture and Differentiation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Embryonic neurospheres were cultured from E13.5 mouse brains as previously described [38 (link)]. Briefly, forebrains were freed of meninges and gently triturated several times in culture medium using a flame-polished Pasteur pipette. Cells from one brain were plated in a 100-mm Petri dish and cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 media (WelGene, Daegu, Korea) supplemented with N-2, B27 supplement (Gibco-Invitrogen, Carlsbad, CA, USA), 20 ng/ml epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF; BD Biosciences, San Jose, CA, USA). EGF and bFGF were added every 2 days. For serial neurosphere formation, primary neurospheres were collected, incubated with Accumax (Millipore Corp, Bedford, MA, USA), and dissociated. For differentiation, dissociated cells were seeded on plates coated with 0.2 mg/ml poly-L-ornithine and 1 µg/ml fibronectin (Sigma, St. Louis, MO, USA) in the absence of growth factors or in the presence of CNTF (BD Biosciences).
7
Capillary-like Tube Formation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Several 24-well culture plates (Sigma-Aldrich) were coated with 100 µL/well of growth factor reduced Matrigel (Becton, Dickinson and Company), and were solidified at 37°C for 1 hour. Before initiation of the tube formation assay, CPC-WT and CPC-KO were washed twice with PBS to remove floating matter and cells, and detached with 0.025% Trypsin-EDTA (Welgene). We have used CPC with or without pre-treatment of serum deprivation culture condition to see the effects of serum deprivation on CPC. Detached CPCs were resuspended with 1 mL serum-containing DMEM/F12 media (Welgene), and counted. 8×104 cells of CPC were plated on growth factor reduced Matrigel coated 24-well culture plates. CPCs were kept in normoxic and hypoxic cultures. We took images of tube formation on a phase-contrast microscope (Olympus Corporation) with a Flex camera (Nikon Corporation, Tokyo, Japan) at the baseline time and 24 hours later.
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!