Cells were grown, transfected and differentiated on Nunc™ Lab-Tek™ II CC2™ Chamber Slide System (ThermoFisher scientific). PLA was carried out with Duolink® In Situ Detection Reagents Orange (Sigma Aldrich) with little modifications from the manufacturer’s instructions, as reported in [20 (link)]. The cells were treated with primary antibodies for anti-α-synuclein (Cell Signaling Technology, 1:100, rabbit) and anti-VDAC1 (Abcam,1:100, mouse) or anti-COXIV (Cell Signaling Technology, 1:100, mouse). Slides were mounted using a minimal volume of Duolink in situ Mounting Medium containing DAPI and images were taken with a Zeiss LSM 710 confocal microscope at 63 × magnification. The microscope settings were kept constant for all images to enable direct comparison. Quantification of signals (number of dots per cell) was obtained from thresholded images using the “analyze particles” feature of ImageJ, which detects isolated continuous objects in the image. Neither PLA puncta size nor circularity was constrained for detection.
Nunc lab tek 2 cc2 chamber slide system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Nunc™ Lab-Tek™ II CC2™ Chamber Slide System is a multi-well cell culture slide system designed for microscopy applications. The system provides a convenient platform for culturing cells and performing various microscopy-based experiments.
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Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (3 mentions)
Crl 6509, by American Type Culture Collection (2 mentions)
Vr 1238, by American Type Culture Collection (2 mentions)
Incubator gas chamber, by Okolab (2 mentions)
Live cell imaging solution, by Thermo Fisher Scientific (2 mentions)
Advanced dmem f12, by Thermo Fisher Scientific (2 mentions)
Anti α synuclein, by Cell Signaling Technology (1 mentions)
Anti vdac1, by Abcam (1 mentions)
Anti cox 4, by Cell Signaling Technology (1 mentions)
Duolink in situ detection reagents orange, by Merck Group (1 mentions)
Evos cell imaging system, by Thermo Fisher Scientific (1 mentions)
Epha2, by Cell Signaling Technology (1 mentions)
Duolink in situ red starter kit mouse rabbit, by Merck Group (1 mentions)
Duolink in situ mounting medium with dapi, by Merck Group (1 mentions)
Wheat germ agglutinin (wga), by Thermo Fisher Scientific (1 mentions)
Wga 647, by Thermo Fisher Scientific (1 mentions)
Image it fixative solution, by Thermo Fisher Scientific (1 mentions)
Wga 488, by Thermo Fisher Scientific (1 mentions)
Ts2r microscope, by Nikon (1 mentions)
13 protocols using nunc lab tek 2 cc2 chamber slide system
1
Proximity Ligation Assay for α-Synuclein Interactions
2
Optimization of KRV Infection Assay
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Subconfluent monolayers of normal rat kidney (NRK) cells (CRL-6509; American Type Culture Collection, Manassas, VA) were grown in DMEM supplemented with 10% FBS on the Nunc Lab-Tek II CC2 Chamber Slide System (Thermo Fisher Scientific) for 24 h. A total of 5 × 104 NRK cells were infected with either KRV or vesicular stomatitis virus (VSV; VR-1238; American Type Culture Collection) at a multiplicity of infection of 1 for 24 h. Cell fixation was performed with 10% neutral buffered formalin, followed by gentle rinsing with 1× PBS at room temperature. Cells were then dehydrated with serial wash steps of ethanol. The fixed, dehydrated slides were stored at −20°C until used in RNAscope ISH assays.
KRV-infected weanling WT and Ifnar1−/− rats were euthanized, and fresh spleens were harvested. Immediately following dissection, tissues were fixed in 10% neutral buffered formalin for 16-32 h at room temperature. Paraffin-embedded sections were sectioned at 5 μm in the UMass Medical School Morphology Core laboratory (www.umassmed.edu/morphology/protocols ).
KRV-infected weanling WT and Ifnar1−/− rats were euthanized, and fresh spleens were harvested. Immediately following dissection, tissues were fixed in 10% neutral buffered formalin for 16-32 h at room temperature. Paraffin-embedded sections were sectioned at 5 μm in the UMass Medical School Morphology Core laboratory (
3
Optimization of KRV Infection Assay
4
Visualizing Cellular Interactions with TNHs and EVs
5
Prostate Organoid Culture Establishment
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Flow-sorted basal and luminal cells were washed with advanced DMEM/F12 (Life Technologies), and resuspended in 10 μl advanced DMEM/F12 and 30 μl Matrigel per well in the Nunc Lab-Tek II CC2 Chamber Slide System (Thermo Fisher Scientific). Chamber slide was put upside down in the 37°C cell culture incubator for 15 min to let the matrigel solidify. Mouse prostate organoid culture medium was prepared according a previous protocol (Drost et al., 2016 (link)). Briefly, the following components were added to advanced DMEM/F12 medium, B27 (50× diluted), HEPES 1 M (100× diluted), GlutaMAX (100× diluted), Penicillin-streptomycin (100× diluted), N-acetylcysteine (1.25 mM), EGF (50 ng/ml), A83-01 (200 nM), Noggin (100 ng/ml), R-spondin 1 (500 ng/ml), DHT (1 nM), Y-27632 dihydrochloride (10 μM). Organoid culture medium was pre-warmed before adding to the wells. The medium was changed with fresh standard medium or paralleled luminal condition medium every 3 days. Organoids were fixed in 4% PFA for 20 min at room temperature before immunofluorescence staining. In situ organoid images were taken using the Keyence microscope in the UCSC Microscopy Shared Facility. Organoid sizes were quantified using ImageJ.
6
Immunocytochemical Characterization of pbMECs
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The epithelial character of the harvested pbMECs was confirmed by immunocytochemistry using the Monoclonal Anti-Cytokeratin, pan-FITC antibody (1:200 in PBS, Sigma-Aldrich, USA, F3418). Approximately 1 × 104 cells were seeded per chamber of Thermo Scientific™ Nunc™ Lab-Tek™ II CC2™ Chamber Slide System and cultured (5% CO2, 37 °C) in the culture media described above until confluency. Cells were first washed three times with PBS and after washing, −20 °C methanol was used for 2 min to fix the cells. Unspecific blocking of the antibody was done with 2% BSA-PBS solution with 30 min incubation at RT. After this step, cells were washed three times with PBS. Antibody solution was added to half of the chambers while PBS was added to the rest of the chambers, whereafter the chambers were incubated at RT and dark for 30 min. After incubation, cells were washed three times with PBS. Imaging was performed with an EVOS Cell Imaging System (Thermo Fisher Scientific, USA) using Evos light cube GFP and light cube DAP.
7
Proximity Ligation Assay for Protein Interactions
8
Fluorescence Microscopy Analysis of EVs
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For the fluorescence microscopy analysis, EVs were labelled with Wheat Germ Agglutinin (WGA) conjugated with Alexa Fluor 647 (WGA647, λEx = 650 nm, Thermo Fisher), ZnO NCs with Atto 550-NHS ester (λEx = 554 nm, ATTO-Tech), and the TNHCD20 nanoconstruct was assembled using the AMCA AffiniPure F(ab′)2 Fragment Goat Anti-Human IgG, Fcγ fragment specific as secondary antibody.
Samples were treated with the same protocol used for the cytofluorimetric analysis and plated in a volume of 100 μL. After 24 and 48 h of culturing at 37 °C, 5% CO2 in 96 well plates, the content of each well was collected, centrifuged, resuspended in 40 μL of the correspondent medium. The 40 μL drop was spotted in a 8-well chamber slide (Thermo Scientific™ Nunc™ Lab-Tek™ II CC2™ Chamber Slide System) and placed at 37 °C, 5% CO2 for 30 min to allow the attachment of the cells. After that, cells were fixed using 250 μL of Image-iT™ Fixative Solution (4% formaldehyde, methanol-free, Thermo Scientific) for 10 min, washed in PBS and cells’ membranes were labelled by incubating cells with 1.25 μL of WGA conjugated with Alexa Fluor 488 (WGA488, λex = 495 nm, Thermo Fisher) for 10 min and washed two other times in PBS. Images were acquired using a wide-field fluorescence-inverted microscope using an immersion oil 100× objective.
Samples were treated with the same protocol used for the cytofluorimetric analysis and plated in a volume of 100 μL. After 24 and 48 h of culturing at 37 °C, 5% CO2 in 96 well plates, the content of each well was collected, centrifuged, resuspended in 40 μL of the correspondent medium. The 40 μL drop was spotted in a 8-well chamber slide (Thermo Scientific™ Nunc™ Lab-Tek™ II CC2™ Chamber Slide System) and placed at 37 °C, 5% CO2 for 30 min to allow the attachment of the cells. After that, cells were fixed using 250 μL of Image-iT™ Fixative Solution (4% formaldehyde, methanol-free, Thermo Scientific) for 10 min, washed in PBS and cells’ membranes were labelled by incubating cells with 1.25 μL of WGA conjugated with Alexa Fluor 488 (WGA488, λex = 495 nm, Thermo Fisher) for 10 min and washed two other times in PBS. Images were acquired using a wide-field fluorescence-inverted microscope using an immersion oil 100× objective.
9
Immunocytochemical Localization of Cytoskeletal Proteins
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LLC-PK1 cells were grown in the 4-well Nunc™ Lab-Tek II CC2™ chamber slide system (Thermo Fisher Scientific, Somerset, NJ, USA; Cat #154917) and treated for 48 h. After treatment, cell morphology was examined under the Nikon (Tokyo, Japan) Ts2R microscope and micrographs were captured. The cell monolayers were then fixed with 4% paraformaldehyde for 15 min at room temperature followed by permeabilization with 0.1% Triton X-100 for 10 min. Monolayers were blocked with 2% w/v bovine serum albumin (BSA) in PBS containing 0.3 M glycine for an hour and then incubated overnight at 4 °C with the primary mouse monoclonal antibodies against α-SMA (1:100), vimentin (1:500) or β-catenin (1:100). Monolayers were then washed with ice-cold PBS, incubated with FITC-labeled anti-mouse IgG antibody (1:1000) for 1 h, then washed thrice with PBS. After the final wash, the slides were mounted in Prolong™ diamond anti-fade reagent with DAPI (Thermo Fisher Scientific, Somerset, NJ, USA; Cat #P36962) and observed under a Nikon (Tokyo, Japan) Ts2R microscope.
10
Visualizing Cellular Interactions with TNHs and EVs
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For in vitro experiments, the KB cells, treated with 15 μg/ml of TNHs and ZnO NCs and with the corresponding number ofEVs, were imaged after 24 h ofincubation at 37°C, 5% CO2. The cells were counted and seeded (3 × 104 cells/well) in 4-well chamber slides (Nunc™ Lab-Tek™ II CC2™ Chamber Slide System, Thermo Fisher Scientific™, MA, USA). After 24 h of growth in standard conditions, the medium was replaced with 500 μl of treatment solution containing the particle for a further 24 h. To maintain healthy cells, in their physiological condition during the acquisition time, an incubator gas chamber (Okolab) equipped with CO2 sensors, temperature unit and an active humidity controller was used. In order to label cell membranes, 2.5 μl of wheat germ agglutinin conjugated with Alexa Fluor 488 dye (λEx = 495 nm) was added to the cell samples and after 10 min of incubation at 37°C, 5% CO2, two washing steps were performed by using 500 μl of live-cell imaging solution (1×, Molecular Probes) at 37°C. ZnO NCs were used as labeled with Atto647 NHS ester dye, EVs were labeled in DiD and in DiO for sample treated with EVs and TNHs, respectively.
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