For the LPS treatment the cells were seeded on cell culture flasks (Falcon, Corning, Inc., Corning, NY, USA) and grown for 24 h. The cells were stimulated with 0.1, 1 or 10 μg/ml highly purified LPS 0111:B4 from E. coli (Sigma-Aldrich, St. Louis, MO, USA) for 16 h before irradiation and after irradiation during the experimental setup with sham-treated cells used as control. Trypan blue (Sigma-Aldrich, St. Louis, MO, USA) staining was used for obviate any toxicity of used LPS doses in our experimental setups.
Cell culture flasks
Manufactured by BD
Sourced in United States
Cell culture flasks are laboratory vessels used for the cultivation and growth of cells in a controlled environment. They provide a sterile, climate-controlled surface for cells to adhere to and proliferate. Cell culture flasks are available in various sizes and shapes to accommodate different cell types and experimental requirements.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Trypan blue, by Merck Group (1 mentions)
Percp cy5.5 anti human cd 105, by BD (1 mentions)
Apc anti human cd 73, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Anti human cd45 pe, by BD (1 mentions)
Anti human hla dr pe, by BD (1 mentions)
Anti human cd11b pe, by BD (1 mentions)
Anti human cd19 pe, by BD (1 mentions)
Anti human cd34 pe, by BD (1 mentions)
Fitc anti human cd90, by BD (1 mentions)
Ficoll gradient solution, by GE Healthcare (1 mentions)
Hank s balanced salt solution, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Cremophore el, by Merck Group (1 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
Texas red dextran, by Thermo Fisher Scientific (1 mentions)
Texas red, by Thermo Fisher Scientific (1 mentions)
4 protocols using cell culture flasks
1
LPS Stimulation and Irradiation Protocol
2
Characterization of hWJ-MSCs by Flow Cytometry
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The hWJ-MSCs that were used were obtained from passage 3 at 80% confluence. The cells were detached from cell culture flasks (Falcon®, Coring, Inc., New York, NY, USA) using trypsin/ethylenediaminetetraacetic acid (EDTA) solution 0.05% (GibcoTM Thermo Fisher Inc., Waltham, MA, USA) and washed with PBS. Then, the hWJ-MSCs were incubated for 30 min at room temperature with saturating concentrations of the following monoclonal antibodies labeled with fluorochromes: PerCP/Cy5.5 anti-human CD 105, FITC anti-human CD90, APC anti-human CD 73, PE anti-human CD 34, PE anti-human CD 45, PE anti-human CD19, PE anti-human CD 11b, PE/Cy7 anti-human HLA-ABC, and PE anti-human HLA-DR (all purchased from BD Biosciences (San Jose, CA, USA). After removing the excess antibody through 3 washes with PBS, the hWJ-MSCs were ana-lyzed according to standard protocols using a flow cytometer (FACS Calibur™, BD Biosciences, San Jose, CA, USA) and FlowJo software (Becton, Dickinson & Company, Franklin, NJ, USA). The cell population was identified, and whether cells were positive or negative for each marker was determined based on the criteria of the International Society for Cell and Genetic Therapy (ISCT).
3
Isolation and Culture of Human BMSCs
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Human BMSCs were isolated from femoral heads of the seven patients following a previously published protocol (Tsai et al., 2015 (link)). In brief, harvested bone marrow was thoroughly mixed with 30 mL of DMEM (Gibco). A syringe with an 18-gauge needle was used to filter out bone debris. Filtered bone marrow was then centrifuged at 1,300 rpm for 5 min to collect cell pellets. After supernatant was removed, a cell pellet was reconstituted in Hank's balanced salt solution (Invitrogen), gently added into a conical tube containing Ficoll gradient solution (GE Healthcare), and centrifuged at 600 × g for 30 min. Mononuclear cells were collected, plated in cell culture flasks (Falcon) with culture medium composed of low-glucose DMEM supplemented with 10% fetal bovine serum (FBS) (Gibco) and antibiotics, and maintained at 37°C in a humidified, 5% CO2 atmosphere. Cells were trypsinized using 0.05% trypsin/EDTA (Gibco) after reaching 70%–80% density confluence and replated at a seeding density of 1,000 cells/cm2. Culture medium was replaced every 3 days.
To study the effects of bone marrow extract on regulation of BMSC activities, we treated BMSCs in culture with or without 300 μg/mL of bone marrow extract for various lengths of time in different experiments. The information of culture time is specified in the section of individual assays.
To study the effects of bone marrow extract on regulation of BMSC activities, we treated BMSCs in culture with or without 300 μg/mL of bone marrow extract for various lengths of time in different experiments. The information of culture time is specified in the section of individual assays.
4
Cellular Uptake of Fluorescent Tracers and Cytotoxic Drugs
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The fluorescent tracers Texas Red (625 Da) and Texas Red dextran (3 kDa) was purchased from Molecular Probes-Life Technologies (Carlsbad, CA). Dulbecco’s modified eagle medium (DMEM) and Fetal bovine serum (FBS) were purchased from Gibco-Life Technologies (Carlsbad, CA). Cell culture flasks were purchased from Falcon (Corning, NY). Radiolabeled (14C)-Paclitaxel (101 mCi/mmol) was purchased from Moravek, Inc. (Brea, CA). Paclitaxel, docetaxel and eribulin was purchased from Selleckchem Chemicals (Houston, TX). Radiolabeled (14C)-Paclitaxel (101 mCi/mmol) was purchased from Moravek, Inc. (Brea, CA). Cresyl violet acetate (0.1%) and Cremophore EL was purchased from Sigma-Aldrich (St. Louis, MO). Anti-GFAP antibody (ab4674) was purchased from abcam (Cambridge, MA). All other chemicals and reagents used were of analytical grade and were used as supplied.
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