Primary mouse and Jurkat T-cells were labelled with 4 µM Fluo-4 AM (Molecular Probes, Invitrogen). For Fluo-4 AM labelling, cells were incubated with the dye for 30 min at room temperature in RPMI (Sigma-Aldrich) without supplements but containing 2.5 mM probenecid. Cells were then washed three times with HBS and finally resuspended in HBS containing 2.5 mM probenecid before addition to the microscope sample container with the prepared microscope coverslip. Cells were imaged at 37°C and 5% CO2 using a 10× air objective on a spinning disk confocal microscope (Zeiss Cell Observer Spinning Disc Confocal), with 488 nm laser excitation and fluorescence detection at ∼530 nm, and with an exposure time of 500 ms and a time between frames of 500 ms for 1000 frames. We used a modified version of CalQuo2 (Lee et al., 2017 (link)), to detect single-cell landing events on the SLB surfaces, and to record fluorescence intensities over time at the coverslip surface. All cells were individually analysed and the fraction of triggering was determined from the total number of cells detected after landing. For the experimental conditions, Ca2+ data were acquired in at least 1000 individual cells over the course of at least three independent experiments.
Cell observer spinning disc confocal
Manufactured by Zeiss
Sourced in Germany
The Cell Observer Spinning Disc Confocal is a high-speed, high-resolution imaging system designed for live-cell microscopy. It utilizes a spinning disc confocal technology to provide optical sectioning and improved image quality. The system is capable of capturing images at high frame rates, making it suitable for monitoring dynamic cellular processes.
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2 protocols using cell observer spinning disc confocal
1
Quantitative Calcium Imaging of T-cells
2
Extracellular Vesicles Modulate Cancer Cell Invasion
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Transwell inserts (Corning, New York, USA) were used in 24-well plates to assess the individual effect of each NOF- and CAF-EV in the cancer cell invasion (HSC-3, SAS, SCC-15, SCC-25) through myogel solidified with low-melting agarose [20 ]. Cells were plated on top of the inserts together with EV in serum-free medium, either individually or pooled into two groups (CAF vs. NOF). After 72 h of incubation, the invasive cells were measured [21 ]. The 3D-myoma organotypic model [22 ] was applied with the HSC-3 cells co-cultured with pooled NOF or CAF derived EV. HSC-3 cells were seeded together with pooled NOF-EV and CAF-EV on the discs and allowed to invade into the myomas for 14 days. After that, the myomas were fixed in 4% formalin solution and immunostained with the pan-cytokeratin antibody (monoclonal mouse anti-human cytokeratin, AE1/AE3–M3515, dilution 1:200, Dako, CA, USA). Pictures were taken from the slides and analyzed on Image J v1.46o (National Institute of Health, USA).
Scratch wound healing assay was used to assess the effect of pooled NOF- and CAF-EV in HSC-3 cell migration, using the Zeiss Cell Observer spinning disc confocal (ZEISS, Ostalbkreis, Germany). Starved cells were seeded in a 24-well plate together with pooled NOF- and CAF-EV and proceed as explained before [20 ].
Scratch wound healing assay was used to assess the effect of pooled NOF- and CAF-EV in HSC-3 cell migration, using the Zeiss Cell Observer spinning disc confocal (ZEISS, Ostalbkreis, Germany). Starved cells were seeded in a 24-well plate together with pooled NOF- and CAF-EV and proceed as explained before [
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