Bone marrow primary monocytes were isolated by flushing femurs and tibias of 8- to 10-week-old C57Bl/6 mice (Charles River Laboratories) with DMEM supplemented with 10% FBS, +1% penicillin/streptomycin, and 1% sodium pyruvate). Aggregates were mechanically dissociated by pipetting, and debris was removed by passaging the suspension through a 70-μm nylon mesh (Corning). Cells were washed twice with complete medium and seeded on 6-well ultra-low attachment surface plates (Corning) Cells were supplemented with 20 ng/mL rmM-CSF (R&D Systems, Minneapolis, MN) and cultured in a humidified incubator at 37 °C and 5% CO2.
6 well ultra low attachment surface plates
Manufactured by Corning
Sourced in United States
The 6-well ultra-low-attachment surface plates are a specialized laboratory equipment designed for cell culture applications. These plates feature a surface that minimizes cell attachment, promoting the formation of spheroid or suspension cultures. The plates are made of high-quality materials and are intended for use in various research and experimental settings.
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Lab products found in correlation
4 protocols using 6 well ultra low attachment surface plates
1
Isolation and Culture of Murine Bone Marrow Monocytes
2
Sphere Formation Assay Protocol
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A total of 5 × 103 cells were plated in 6-well ultralow attachment surface plates (Corning Life Science). EGF (20 ng/ml, Invitrogen), bFGF (10 ng/ml, Invitrogen), and 2% B27 (Invitrogen, Carlsbad, CA) were formulated in a medium. After 2 weeks, the number of spheroids was counted manually by a microscope.
3
Mammosphere Formation and Ancistrobrevolines Effects
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Mammospheres derived from MCF-7 cells were formed by seeding the cells in 6-well ultra-low-attachment surface plates (Corning, Tewksbury, MA, USA) at a density of 2 × 104 cells/well and cultured in serum-free DMEM-Ham's F12 nutrient mixture (1 : 1, v/v), supplemented with 5 mg mL−1 insulin, 0.5 mg mL−1 hydrocortisone, 2% B-27 supplements, 10 ng mL−1 fibroblast growth factor, and 20 ng mL−1 epidermal growth factor. The cells were incubated at 37 °C and 5% CO2 for 5 d to form mammospheres.69 (link) To study the effect of ancistrobrevolines A (14) and B (15) on mammosphere formation, the cells were treated with different concentrations (10, 30, 50, 70, and 100 μM) of the agents and then allowed to form spheres for the next 5 d. The compound solutions were prepared using DMSO with its final non-toxic concentration (0.1%). An equal amount of DMSO was added to the non-treated control group. After 5 d, the mammospheres were photographed using phase-contrast microscopy, and the images were analyzed using the ImageJ analysis software.69 (link)
4
Colonosphere Formation and Rutin Effects
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Colonosphere (colon cancer stem-like cells) formation mimics the colon cancer stem cells’ properties and has been used to study the molecular mechanism of the anti-colon cancer stem cell property of lead molecules [50 (link)]. The colon cancer cell line (HCT-116) was utilized to form colonospheres. A total of 2 × 104 cells/well were seeded in 6-well ultra-low-attachment surface plates (Corning, New York, NY, USA). The cells were cultured in serum-free DMEM-F12 (1:1, v/v) supplemented with the necessary ingredients described in previous studies [48 (link)]. After that, the cells were incubated in a humid environment (37 °C, 5% CO2) for five days to allow the formation of colonospheres [51 (link)]. To study the effect of rutin on colonosphere formation, the cells were pre-treated at IC30 and IC50 concentrations (obtained in the MTT assay in HCT-116 cells). Similarly, the vehicle-treated HCT-116 cells were allowed to form colonospheres and considered as the control group. For the experiments, rutin was dissolved in DMSO, thus an equal amount of DMSO was added to vehicle-treated colonosphere group. Following this, the Notch-1 siRNA and siRNA-control transfected HCT-116 cells were incubated for five days and allowed to form colonospheres. After the incubation the colonospheres from the test groups were photographed using microscopy. The images were assessed through Image J software.
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