The 2.5 × 104 cells non stimulated (ctrl) and 0.1 mg/mL 5-FU, 0.1 mg/mL 5-FU + 0.3 µg/mL F6 and 0.3 µg/mL F6 as single agent respectively, were placed in 500 μL DMEM + 0.1% FBS medium (DMEM F12 + 0.1% horse serum + 10 ng/mL TGF-β1 in case of MCF-10A cells) in the upper side of 8-µm filters (BD Bio-CoatTM growth factor reduced MATRIGELTM invasion chamber, BD Biosciences-Discovery Labware, Two Oak Park, Bedford, MA, USA) (upper chamber) and placed in wells of a 24-well plate (Falcon, BD Biosciences) (lower chamber), containing 0.8 mL of DMEM 10% FBS medium (DMEM F12 + 10% horse serum in case of MCF-10A cells). After 24 h of incubation, the invasive cells on the lower surface of membranes were fixed, stained with Hemacolor® (HX54775574, Merck, Darmstadt, Germany) and examined microscopically. Cellular invasion was determined by counting the number of cells on membranes in at least 4–5 randomly selected fields using a Zeiss Axiovert 10 optical microscope. For each data point, four independent experiments in duplicate were performed.
Axiovert 10 optical microscope
Manufactured by Zeiss
Sourced in Germany
The Axiovert 10 is an optical microscope designed for basic imaging applications. It features a compound configuration, providing magnification and high-resolution visualization of samples. The microscope utilizes transmitted light illumination and can accommodate a variety of objective lenses for different magnification levels.
Automatically generated - may contain errors
4 protocols using axiovert 10 optical microscope
1
Invasion Assay of Breast Cancer Cells
2
Cell Migration Assay under Microgravity
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Cells were treated for 24 hours in μg or kept at 1g then were placed (5 × 103 cells) in the upper side of 8-μm filters (Falcon, BD Biosciences) (upper chamber) in 0.5 ml DMEM 0.1% FBS. These filters were placed in a 24-well plate (Falcon, BD Biosciences, San Jose, CA, USA) (lower chamber), containing 0.8 ml DMEM 10% FBS. Chambers were kept in an incubator for 24 hours. After incubation, cells from the upper surface of filters were removed and the migratory cells on the lower surface of membranes were fixed and stained with hematoxylin/eosin. The membranes were examined microscopically, and cell migration was determined by counting the number of cells on membranes in at least 4–5 randomly selected fields 10X magnification using a Zeiss Axiovert 10 optical microscope. Cellular migration value was reported as mean ± standard deviation (SD) of migrating cells/field count and normalized to control cells. For each experimental conditions, four independent experiments in duplicate were performed.
3
Evaluating Cell Migration Inhibition
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The 2.5 × 104 cells non-stimulated (ctrl) and stimulated 0.1 mg/mL 5-FU, 0.1 mg/mL 5-FU + 0.3µg/mL F6 and 0.3µg/mL F6 respectively, were placed in 500 μL DMEM + 0.1% FBS medium (DMEM F12 + 0.1% horse serum + 10ng/mL TGF-β1 in case of MCF-10A cells) in the upper side of 8-µm filters (Falcon, BD Biosciences, San Jose, CA, USA (upper chamber) and placed in wells of a 24-well plate (Falcon, BD Biosciences) (lower chamber), containing 0.8 mL of DMEM + 10% FBS medium (DMEM F12 + 10% horse serum in case of MCF-10A cells). After 24 h of incubation, the migratory cells on the lower surface of membranes were fixed, stained with Hemacolor® (HX54775574, Merck, Darmstadt, Germany) and examined microscopically cellular migration was determined by counting the number of cells on membranes in at least 4–5 randomly selected fields using a Zeiss Axiovert 10 optical microscope. For each data point, four independent experiments in duplicate were performed.
4
Simulated Microgravity Impacts Cell Migration
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Human dermal fibroblasts, either exposed to simulated microgravity in the RPM or cultured in normal gravity (1 g), were placed (5 × 103 cells) in DMEM 0.1% FBS in the upper side of 8-μm filters (Falcon, BD Biosciences) for the migration assay or in Matrigel-coated 8 μm filters (BD Bio-Coat™ growth factor reduced MATRIGEL™ invasion chamber, Falcon, BD Biosciences) for the invasion assay. They were then placed in the wells of a 24-well plate (Falcon, BD Biosciences), containing 0.8 mL of DMEM 10% FBS. After 24 h of incubation at 37 °C, cells from the upper surface of the filters were removed with gentle swabbing, and cells that had migrated to or invaded across the Matrigel’s lower surface were fixed and stained with hematoxylin/eosin. The membranes were examined microscopically to determine cellular migration and invasion by cell counting in at least 4–5 randomly selected fields for each. A Zeiss Axiovert 10 optical microscope was used. For each data point, four independent experiments in duplicate were performed.
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