In order to evaluate granule polarization during NK cell degranulation, the surface distribution of CD107a antigen was analysed by confocal microscopy and ImageStream. For confocal microscopy observations, a small aliquot (10 µL) of the same samples prepared for flow cytometric analysis (a 2h assay time) was used. Samples, placed on a polylysinated slide, were observed by a Leica TCS-SP5 confocal microscope equipped with DMI 6000 CS inverted microscope (Leica Microsystems CMS GmbH, Mannheim, Germany) and analysed with the Leica Applications Suite Advanced Fluorescence (LAS AF) software. The surface distribution of CD107a was also evaluated on NK cells incubated with Jurkat cells (NK granule polarization positive control). This cell line is a NK sensitive target [[54 (link)]] able to activate NK cytotoxic response, thus inducing a contextual polarization of NK granules, necessary for an effective cytolytic activity. To confirm confocal microscopy data, some experiments were performed with an ImageStream device (Luminex, Austin, TX, USA) equipped with a 488 nm laser. ImageStream data were analysed with IDEAS 3.0 software (Amnis Corporation, Seattle, WA, USA).
Dmi 6000 cs inverted microscope
Manufactured by Leica Microsystems
Sourced in Germany
The Leica DMI 6000 CS is an inverted microscope designed for advanced cell and tissue imaging. It features a stable and precise optical system, enabling high-resolution visualization of samples. The microscope is equipped with a variety of illumination options and can accommodate a range of objectives to suit different imaging requirements.
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7 protocols using dmi 6000 cs inverted microscope
1
Confocal and Imagestream Analysis of NK Cell Degranulation
2
TUNEL Assay for Apoptosis Detection
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Control and treated cells, directly processed on coverslips in Petri dishes, were washed and fixed with 4% paraformaldehyde in PBS, pH 7.4, for 30 min, rinsed with PBS and permeabilized with a 2:1 mixture of ethanol and acetic acid for 5 min at -20°C. For the TUNEL technique, all reagents were part of the ApopTag PlusR kit (D.B.A., Dallas, TX, USA) and the procedures were carried out according to the manufacturer’s instructions, as described by Salucci et al.14 (link) Specimens were observed with a Leica TCS-SP5 connected to a DMI 6000 CS Inverted Microscope (Leica Microsystems CMS GmbH, Mannheim, Germany); excitation was at 488 nm and emission signals were detected at 517 nm.
3
Apoptosis and Necrosis Cell Imaging
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Cells, fixed with 4% paraformaldehyde in PBS pH 7.4 for 30 min and deposited on poly-lysinated coverslips in Petri dishes, were washed twice using PBS. Cells were pre-treated with RNasi A 10 μg/mL in PBS for 30 min and then exposed to an equal mixture of PI (1 μg/mL; Life Technologies) and AO (1 μg/mL; Life Technologies) diluted in PBS at room temperature in the dark for 10 min.
Specimens were observed with a Leica TCS-SP5 CLSM connected to a DMI 6000 CS Inverted Microscope (Leica Microsystems CMS GmbH; FICT and PI excitation were at 488 and 500 nm, respectively, and their emission signals were detected at 617 and 525.) CLSM Images are presented as maximum intensity projection or single-plane images.
AO and PI are intercalating nucleic acid specific fluorochromes which emit green and red fluorescences, respectively, when they are bound to DNA. Only AO can diffuse through the plasma membrane of viable and early apoptotic cells. Viable cells show green nucleus with intact structure while apoptotic cells exhibit a bright-green nucleus showing condensation of chromatin in dense green areas. Late apoptotic and necrotic cells are stained with both AO and PI. Comparatively, PI produces the highest intensity emission in necrotic cells. Hence, late apoptotic cells exhibit an orange nucleus showing condensation of chromatin, whereas necrotic cells display a red nucleus [43 (link)].
Specimens were observed with a Leica TCS-SP5 CLSM connected to a DMI 6000 CS Inverted Microscope (Leica Microsystems CMS GmbH; FICT and PI excitation were at 488 and 500 nm, respectively, and their emission signals were detected at 617 and 525.) CLSM Images are presented as maximum intensity projection or single-plane images.
AO and PI are intercalating nucleic acid specific fluorochromes which emit green and red fluorescences, respectively, when they are bound to DNA. Only AO can diffuse through the plasma membrane of viable and early apoptotic cells. Viable cells show green nucleus with intact structure while apoptotic cells exhibit a bright-green nucleus showing condensation of chromatin in dense green areas. Late apoptotic and necrotic cells are stained with both AO and PI. Comparatively, PI produces the highest intensity emission in necrotic cells. Hence, late apoptotic cells exhibit an orange nucleus showing condensation of chromatin, whereas necrotic cells display a red nucleus [43 (link)].
4
Apoptosis Detection via TUNEL Assay
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Control and treated cells were fixed with 4% paraformaldehyde in phosphate buffer saline (PBS) pH 7.4 for 30 min, then deposited on poly-lysinated coverslips in Petri dishes overnight at 4°C. After PBS washing, samples were permeabilized with a 2:1 mixture of ethanol and acetic acid for 5 min at -20°C.21 (link) For the TUNEL technique, all reagents were part of the Apoptag Plus kit (D.B.A., Oncor, Dallas, TX, USA) and procedures were carried out according to the manufacturer’s instructions and as described in Salucci et al.20 (link) Finally, slides were mounted with an antifading medium.22 (link) Specimens were observed with a Leica TCS-SP5 confocal laser scanning microscope (CLSM) connected to a DMI 6000 CS inverted microscope (Leica Microsystems CMS GmbH, Mannheim, Germany); excitation was at 488 nm and emission signals were detected at 517 nm.
5
TUNEL Assay for Apoptosis Detection
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Control and treated cells were fixed with 4% paraformaldehyde in phosphate buffer saline (PBS) pH 7.4 for 30 min. They were then deposited on poly-lysinated coverslips in Petri dishes overnight at 4 °C. All samples were rinsed with PBS and permeabilized with a 2:1 mixture of ethanol and acetic acid for 5 min at −20 °C. For the TUNEL technique, all reagents were part of the Apoptag Plus kit (D.B.A., Oncor, Dallas, TX, USA) and procedures were carried out according to the manufacturer’s instructions. Cells were treated with TdT buffer for 10 min at room temperature and incubated with the reaction buffer containing the TdT enzyme, for 1 h at 37 °C in a humidified chamber. The reaction was blocked using the stop buffer for 10 min. Cells were incubated with a FITC-conjugated anti-digoxigenin antibody for 30 min at room temperature. Finally, slides were mounted with an antifading medium [42 (link)]. Specimens were observed with a Leica TCS-SP5 confocal laser scanning microscope (CLSM) connected to a DMI 6000 CS inverted microscope (Leica Microsystems CMS GmbH, Mannheim, Germany); excitation was at 488 nm and emission signals were detected at 517 nm. CLSM Images are presented as maximum intensity projection or single-plane images.
6
Fluorescent Imaging of Dendritic Granules
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Neurons were transfected with scFv-sfGFP-GB1 and 24xV4-ODC-Arc 3'UTR. They were fixed with 3.7% formaldehyde in PBS for 10 min, washed with PBS, and then, the specimens were mounted in Mowiol. Fluorescence images were acquired using a TCS SP8 MP confocal microscope equipped with a DMI6000CS inverted microscope (Leica Microsystems, Wetzlar, Germany) and an HC PL APO 63 × /1.20 W CORR CS2 water immersion objective. The GFP fluorescence in the dendritic granules was measured and normalized by min-max normalization in each neuron. In these analyses, we noted that fluorescence images of co-expressed Sirius-tagged RBPs were noisy due to the laser wavelength (405 nm) used for Sirius (excitation peak: 355 nm).
7
Mitochondrial Membrane Integrity Evaluation
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To monitor mitochondrial behavior, fresh cells were treated with 10 nonyl-acridine orange (NAO) and JC1 probe for evaluating mitochondria membrane integrity and potential membrane functionality, respectively. In particular, NAO binds cardiolipin, a phospholipid located in the inner mitochondrial membrane permitting to identify peroxidation events. Fresh cells were exposed to 50 nM NAO for 10 min at room temperature or to JC1 for 20 min at 37°C.23 Samples were observed through a Leica TCS-SP5 Confocal connected to a DMI 6000 CS Inverted Microscope (Leica Microsystems CMS GmbH; objectives 40x and 60x); excitation was at 488nm (FITC and NAO); emission signals were detected at 519 nm (NAO) or 525 nm (JC1). CLSM images are presented as single-plane images or Z-stack projections.24 (link)
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