For microscopy, cells were transferred to a microscopy slide coated with a thin film of 1% (w/vol) agarose (Roth). Fluorescence microscopy was carried out at room temperature using a Zeiss Axio Observer Z1 microscope (Zeiss, Jena, Germany) equipped with an HXP 120 V light source (Zeiss, Jena, Germany), an αPlan-APOCHROMAT 100×/1.46 oil immersion objective (Zeiss, Jena, Germany) and an Axio Cam MR3 camera (Zeiss, Jena, Germany). Visualization of mCherry was achieved using Carl Zeiss filter set 64 HE (574–599 nm excitation, 605 nm beam splitter and 612–682 nm emission). Image acquisition and analysis were performed with Zen 2 software (Zeiss, Jena, Germany).
α plan apochromat 100 1.46 oil immersion objective
Manufactured by Zeiss
Sourced in Germany
The ZEISS α Plan-Apochromat 100×/1.46 oil immersion objective is a high-magnification objective lens designed for use in microscopy applications. It provides a numerical aperture of 1.46 and a magnification of 100x, making it suitable for high-resolution imaging and analysis of fine details in samples.
Automatically generated - may contain errors
Lab products found in correlation
Cellmask deep red, by Thermo Fisher Scientific (2 mentions)
Micropatterned glass bottom of 35 mm dishes, by MatTek (2 mentions)
Elyra s1 microscope, by Zeiss (2 mentions)
Cytofix, by BD (2 mentions)
Zen 2, by Zeiss (1 mentions)
Hxp 120 5, by Zeiss (1 mentions)
Axiocammr3 camera, by Zeiss (1 mentions)
Axio observer z1 microscope, by Zeiss (1 mentions)
Cell observer sd, by Zeiss (1 mentions)
Ixon3 885, by Oxford Instruments (1 mentions)
4 protocols using α plan apochromat 100 1.46 oil immersion objective
1
Fluorescence Microscopy of mCherry-Labeled Cells
2
Spinning Disk Confocal Microscopy for Vesicle Dynamics
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The Cell Observer SD spinning disk confocal microscope
(Zeiss, Germany) was used for vesicle recording. It was equipped with
an α Plan-Apochromat 100×/1.46 oil immersion objective
(Zeiss, Germany). 512 × 512 pixel images were recorded with an
EMCCD camera (iXon3885, Andor, UK) using 2 × 2 binning with a
0.133 μm pixel size at a rate of 33 frames per second (fps)
with a video integration time of 30 ms. At least 5000 images were
recorded for each of the vesicles. Samples were illuminated with a
488 nm laser, and emitted light passed through the 527/54 filter.
All samples were measured at 22 ± 1 °C (295 K). All measurements
have been performed in a dedicated PTFE observation chamber with very
limited height (equal to 300 μm) to reduce the effect of uncontrolled
vesicle movements. The value of depth of focus was equal to 0.85 μm.
To improve further quality of the analysis, the radius of the vesicle
was calculated for each image, and when the fluctuations of the radius
were unacceptable, as a result of misdetection caused by noise or
other reasons described in previous work,19 (link) the image in the series was discarded from further analysis.
(Zeiss, Germany) was used for vesicle recording. It was equipped with
an α Plan-Apochromat 100×/1.46 oil immersion objective
(Zeiss, Germany). 512 × 512 pixel images were recorded with an
EMCCD camera (iXon3885, Andor, UK) using 2 × 2 binning with a
0.133 μm pixel size at a rate of 33 frames per second (fps)
with a video integration time of 30 ms. At least 5000 images were
recorded for each of the vesicles. Samples were illuminated with a
488 nm laser, and emitted light passed through the 527/54 filter.
All samples were measured at 22 ± 1 °C (295 K). All measurements
have been performed in a dedicated PTFE observation chamber with very
limited height (equal to 300 μm) to reduce the effect of uncontrolled
vesicle movements. The value of depth of focus was equal to 0.85 μm.
To improve further quality of the analysis, the radius of the vesicle
was calculated for each image, and when the fluctuations of the radius
were unacceptable, as a result of misdetection caused by noise or
other reasons described in previous work,19 (link) the image in the series was discarded from further analysis.
3
Characterizing NK Cell Membrane-Surface Interactions
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NK cells were seeded on the micropatterned glass bottom of 35-mm dishes (MatTek) at 5 × 10 5 cells/ml and left to interact for 30 minutes. NK cell outer membranes were then stained with CellMask Deep Red (Thermo Fisher Scientific) at 10 µg/mL for 10 minutes, washed with PBS before 10-minute fixation with Cytofix (BD Biosciences) containing 4% paraformaldehyde. The samples were then blocked using PBS with 5% w/v BSA and finally mounted for immediate imaging. Images were acquired using an Elyra S1 microscope equipped with an α Plan-Apochromat 100×/1.46 oil immersion objective (Carl Zeiss AG). NK cells having spread symmetrically over the entire AIS were selected by widefield imaging before switching to TIRF mode where the imaging focus plane was set according to the AIS. To assess if NK cells' plasma membrane were in close proximity to the surface across the AIS, radial fluorescence intensity profiles of the NK cell membrane over the prints were generated using the Radial Profile plugin in ImageJ (NIH). The resulting curves were normalized in radius (0 to 1) and in intensity (integrated fluorescence intensity set to 1) to account for individual staining differences.
4
Characterizing NK Cell Membrane-Surface Interactions
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+ Expand
NK cells were seeded on the micropatterned glass bottom of 35-mm dishes (MatTek) at 5 × 10 5 cells/ml and left to interact for 30 minutes. NK cell outer membranes were then stained with CellMask Deep Red (Thermo Fisher Scientific) at 10 µg/mL for 10 minutes, washed with PBS before 10-minute fixation with Cytofix (BD Biosciences) containing 4% paraformaldehyde. The samples were then blocked using PBS with 5% w/v BSA and finally mounted for immediate imaging. Images were acquired using an Elyra S1 microscope equipped with an α Plan-Apochromat 100×/1.46 oil immersion objective (Carl Zeiss AG). NK cells having spread symmetrically over the entire AIS were selected by widefield imaging before switching to TIRF mode where the imaging focus plane was set according to the AIS. To assess if NK cells' plasma membrane were in close proximity to the surface across the AIS, radial fluorescence intensity profiles of the NK cell membrane over the prints were generated using the Radial Profile plugin in ImageJ (NIH). The resulting curves were normalized in radius (0 to 1) and in intensity (integrated fluorescence intensity set to 1) to account for individual staining differences.
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