A subset of the isolated primary osteocytes was seeded in collagen-coated coverslips mounted in the Focht Chamber System 2 (FCS2) (Bioptechs, Butler, PA). The cells where subjected to flow shear stress (FSS) at 4 dynes/cm2 for 15 min in the presence of 25 μM ethidium bromide (EtBr) in the recording media (HCO3− -free αMEM medium buffered with 10 mM HEPES). The FSS chamber was placed on the stage of a Nikon eclipse TE 200 microscope. The fluorescence images were captured every 30 sec at the excitation wavelength of 561 nm. The intensity of EtBr fluorescence in cells was measured and quantified by Image J software. Primary osteocytes were microinjected using an Eppendorf micromanipulator InjectManNI 2 and Femtojet (Eppendorf) at 37°C with 10 mM Alexa594 dye in PBS for 5 min to examine gap junction cell coupling, which was observed under an inverted microscope equipped with Lambda DG4 device (Sutter instrument CO, Novato CA) with mercury arc lamp illumination and a Nikon eclipse (Nikon, Japan) using a rhodamine filter.
Focht chamber system 2
Manufactured by Bioptechs
Sourced in United States
The Focht Chamber System 2 (FCS2) is a versatile environmental control system designed for live cell imaging applications. It provides precise control over the temperature, humidity, and gas composition within the imaging chamber. The FCS2 is compatible with a variety of microscope systems and can be used to maintain optimal conditions for live cell experiments.
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Lab products found in correlation
3 protocols using focht chamber system 2
1
Osteocyte Mechanosensing under Flow Stress
2
Inhibition of CTC Arrest by P-Lipo
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To investigate the inhibition effect of P-Lipo on the CTC arrest by endothelial cells, Focht Chamber System 2 (FCS2®, Bioptechs Inc, USA) was built for flow cell imaging on inverted microscopes. To mimic the flowing CTC arrest in distant capillary beds, 50 × 106 HUVECs were seeded in the lower coverslip and were exposed to the fluid flow containing 10 ng/mL of TNF-α for 12 h. 4T1-GFP cells were collected using trypsin digestion and resuspended in HBSS. Afterward, 4T1-GFP (1 × 104/mL) and P-Lipo (or Lipo, 20 μg/mL) were filled into the syringes and connected to the FCS2 chamber via a three-way valve. A constant flow rate was generated by a syringe pump. Once the flow was generated, a high-speed video of moving 4T1-GFP cells was recorded using a trinocular inverted fluorescence phase-contrast microscope (Olympusix71, Japan). After 10 min perfusion, the picture of 4T1-GFP latched onto HUVECs was also gathered and the number of 4T1-GFP was analyzed using ImageJ.
3
DAPI Staining and Flow Chamber Setup
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Nuclear staining and flow chamber assembly were performed as described previously [25 (link)]. Fixed embryos on round cover slips were stained with DAPI in 2× SCC (1:1000, Thermo Fisher) for 5–10min immediately prior to imaging and washed 3 times with 2× SCC for 5–10min.
Specimens were mounted using a flow cell (Focht Chamber System 2 (FCS2®), Bioptechs), equipped with a micro aqueduct and attached to a home-build fluidic system [68 (link)] attached to the microscope. During flow cell assembly, 0.1µm Tetraspeck (ThermoFisher) beads in 2× SCC were allowed to adhere to the coverslip in order to track sample drift during image acquisition.
Specimens were mounted using a flow cell (Focht Chamber System 2 (FCS2®), Bioptechs), equipped with a micro aqueduct and attached to a home-build fluidic system [68 (link)] attached to the microscope. During flow cell assembly, 0.1µm Tetraspeck (ThermoFisher) beads in 2× SCC were allowed to adhere to the coverslip in order to track sample drift during image acquisition.
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