A single suspension of bone marrow-derived all nucleated cells (ANCs) from mouse femurs and tibias was seeded at a density of 2.5×105 cells per square centimeter on 10 cm culture dish (Corning, NY, USA) at 37°C in 5% CO2. Non-adherent cells were removed after 48 hours and attached cells were maintained for 16 days in alpha minimum essential medium (α-MEM, Invitrogen) supplemented with 20% fetal bovine serum (FBS, Equitech-Bio, Kerrville, TX, USA), 2 mM L-glutamine, 55 μM 2-mercaptoethanol, 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen). Colonies forming attached cells were passed once for further experimental use.
10 cm culture dish
Manufactured by Corning
Sourced in United States
The 10 cm culture dish is a laboratory equipment item designed for cell culture applications. It provides a standard-sized, sterile container for the growth and maintenance of cells in a controlled environment.
Automatically generated - may contain errors
Lab products found in correlation
α mem, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Equitech-Bio (1 mentions)
Facsverse, by BD (1 mentions)
Facsuite software, by BD (1 mentions)
Bovine serum albumin (bsa), by Fujifilm (1 mentions)
Anti human cd73, by BioLegend (1 mentions)
Accutase, by Nacalai Tesque (1 mentions)
Polyethylenimine (pei), by Polysciences (1 mentions)
Pspax2, by Addgene (1 mentions)
Pmd2 g, by Addgene (1 mentions)
Guava easycyte system, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Isotype matched mouse monoclonal antibodies, by BD (1 mentions)
Trypsin edta, by Corning (1 mentions)
Human transferrin, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Corning (1 mentions)
Caco 2, by Merck Group (1 mentions)
8 protocols using 10 cm culture dish
1
Expansion of Mouse Bone Marrow Cells
2
Immunophenotyping of Mesenchymal Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were cultured in a 10-cm culture dish (Corning) at 70% confluency, and treated with accutase (Nacalai Tesque, Kyoto, Japan) to detach them from the culture dish. The cells were then fixed with 2% paraformaldehyde in PBS for 15 min, and blocked with 1% BSA (Wako) in PBS for 20 min. Subsequently, the cells were incubated (1 × 106 cells per 100 μL) with PE-labeled anti-human CD90 (#BL328110, BioLegend, CA, USA), anti-human CD73 (#BL344004, BioLegend), anti-human CD34 (#BL343506, BioLegend), and mouse IgG1 kappa isotype control antibodies (#BL400114, BioLegend) for 1 h at room temperature in dark. Populations of 10,000 cells were analyzed using FACSVerse (BD Bioscience, CA, USA) and FACSuite software (BD Bioscience).
3
Lentiviral Vector Production in HEK293T
4
Isolation and Characterization of Murine Bone Marrow-Derived Mesenchymal Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The proximal and distal metaphysis of the femurs and tibias was cut and the bone marrow was flushed from the diaphysis with 1% FBS (Gibco, USA) in PBS. Single-cell suspension of all nuclear cells was seeded at a density of 2 × 105 cells per square centimetre on 10-cm culture dish (Corning, NY, USA) at 37°C in 5% CO2. Non-adherent cells were removed after 24 hours, and the attached cells were maintained BM-MSCs. Surface antigens of BM-MSCs were analysed using flow cytometry. The monoclonal antibodies used (BD Biosciences) were CD44-FITC, CD29-PE, Sca-1-PE, CD34-APC, CD45-FITC and CD11b-PE. The cells were incubated with 1% bovine serum albumin (BSA; Gibco) for 30 minutes to block non-specific antigens. Following two washes in PBS, the cells were resuspended and analysed using the Guava easyCyte™ system (Millipore, Billerica, MA, USA). Non-Specific fluorescence was determined with isotype-matched mouse monoclonal antibodies (BD Biosciences).
5
Isolation and Expansion of Human Amniotic Fluid Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples of amniotic fluid (AF) were obtained from Suzhou Hospital Affiliated with Nanjing Medical University following routine amniocentesis carried out on pregnant women after 19-22 weeks of gestation. All procedures were performed following the guidelines established by Suzhou Hospital Affiliated with Nanjing Medical University Ethics Boards. Written consent was obtained from each woman after informing her that the amniotic fluid would be used for both genetic analysis and research purposes. After amniocentesis, immunoselection with an antibody specific for human c-Kit (CD117) was used to isolate AFS cells12 (link). The cells were isolated from each AF sample and then plated into a 10 cm culture dish (Corning) and expanded. The total cell count in 5 ml of amniotic fluid amounted to approximately 1 × 106, of which approximately 1 × 104 were hAFS cells. Approximately 95% of the non-adherent cells were removed after 24 h, and the culture media was replaced every day. Cells were passaged by trypsinization (0.25% trypsin, 0.1% EDTA) and expanded serially with a split ratio of 1:3 at 70% confluence in 2 or 3 days. Cultures of hAFS cells were maintained in a humidified incubator under 5% CO2 at 37 °C.
6
Culturing Caco-2, Raw264.7, and HBEpiC Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Caco-2 and Raw264.7 cell lines, purchased from Bioresource Collection and the Research Center (BCRB; Hsinchu, Taiwan), were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Corning, NY, USA) containing 10% inactive Fetal Bovine Serum (FBS, Corning) and 1 mM pyruvate (Corning). Caco-2 cells were supplemented with 0.01 mg/mL human transferrin (Sigma-Aldrich Chemical Co.). The human bronchial epithelial (HBEpiC, Cat:3210) cell line was purchased from ScienCell (Carlsbad, CA, USA) and maintained in Bronchial Epithelial Cell Medium (BEpiCM, Cat:3211, ScienCell). All cell lines were cultured in a 10 cm culture dish (Corning) in a humidified incubator at 37 °C with 5% CO2. The cells were harvested using 0.25% Trypsin/EDTA (Corning) when they reached 85−90% confluence.
7
Quantifying Mesenchymal Stem Cell Colonies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The CFU-F assay was used to enumerate colonyforming cells (MSCs), as reported previously (El-Jawhari et al., 2017) . Equal volumes (200 µL) of paired BMA and BMAC samples were treated with RBC lysis buffer. Then, cells were seeded in 10 cm culture dish (Corning). Each seeded sample contained an average of 5.3 × 10 6 (1.2-7.4 × 10 6 ) and 21 × 10 6 (3-38 × 10 6 ) cells for BMA and BMAC, respectively. All cell cultures were maintained in StemMACS™ MSC Expansion Media (Miltenyi Biotec), supplemented with 1 : 100 penicillin/streptomycin (Sigma-Aldrich), at 37 °C and 5 % CO 2 for 14 d with half-medium changes twice weekly. For colony counting, the medium was removed and the colonies were fixed using 10 % formalin (Sigma-Aldrich). Then, methylene blue dye (Sigma-Aldrich) was used to visualise colonies. The colonies were counted, and the normalised count per 1 mL of BMA or BMAC was calculated. For assessing the proliferative capacity of native MSCs, colonies were scanned and analysed using Image J software (NIH). The coloured RGB images of CFU-F colonies were analysed as binary 8-bit greyscale images and the colony integrated density was calculated as area × mean grey density value.
8
Lentiviral Transduction of 293T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Of 293T cells for lentivirus production were seeded in a 10 cm culture dish (Corning) until 80% confluence. Plasmids with the proper proportion (fasl and tert gene expression vector: psPAX: pCMV-VSV-G (all from Addgene) = 5:3:2) were mixed in opti-MEM (Invitrogen) with Lipofectamin LTX (Invitrogen) according to the protocol of the manufacturer. EGFP expression plasmid (Addgene) was used as a control. The supernatant was collected at 48 h after transfection and filtered through a 0.45 μm filter to remove cell debris. For infection, the supernatant containing lentivirus was added into the target cell culture in the presence of 4 μg/ml polybrene (SIGMA), and the transgene expression was validated by GFP under microscopic observation.
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!