Morphology of the cells, after the three-day direct contact with the powder specimens, was evaluated via immunofluorescent imaging (IF). DAPI was used to label the cell nuclei, while Alexa Fluor 594-conjugated phalloidin was used to visualize cytoskeleton f-actin filaments. Briefly, samples were washed with PBS and fixed for 20 min with 4% paraformaldehyde. Subsequently, they were washed twice with PBS to elute the fixative and sample powder residues, and then stained with a mixture of DAPI and phalloidin in 0.5% Tween®-20 for one hour. Stained samples were checked using the digital light microscope (Invitrogen EVOS Floid, from Thermo Scientific, Waltham, MA, USA).
Evos floid
Manufactured by Thermo Fisher Scientific
Sourced in United States
The EVOS Floid is a compact and portable inverted fluorescence microscope designed for live-cell imaging. It provides a stable and controlled environment for observing cells and tissues under a variety of conditions.
Automatically generated - may contain errors
5 protocols using evos floid
1
Cell Morphology Evaluation via Immunofluorescence
2
Mitochondrial Membrane Potential Assay
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To measure mitochondrial membrane potential, G3 MB cells were loaded with the JC-1 dye (Cayman Chemicals) according to manufacturer’s instructions. Cells were suspended in culture medium containing a final concentration of 2 μM JC-1 dye and incubated at 37 °C for 15 minutes protected from light. Following incubation cells were washed and imaged in a 96-well black-walled plate using an EVOS Floid (ThermoFisher) fluorescent microscope. JC-1 aggregates were imaged at ex/em of 535/595 nm and JC-1 monomers were imaged at ex/em of 485/535 nm. Fluorescent intensity was quantified using Image J software and the ratio of the fluorescent intensity of JC-1 aggregates to monomers was calculated.
3
Cell Viability Assay for Biomaterials
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In this assay, L929 cells were seeded into 24-well plates alongside previously placed sterile test biomaterials. In this method, the direct interaction between the cells and the test material was checked by assessing the morphology of the cells and the percentage of live and dead cells (Cell Viability Imaging Kit, Green/Red), which was evaluated on a fluorescence microscope EVOS FLoid (Thermo Scientific, Waltham, MA, USA).
4
Apoptosis Assessment by Multiple Assays
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Apoptosis was investigated by three different assays: Annexin V/7-AAD flow cytometry assay, terminal-deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) assay and western blot analysis of caspases expression and cleavage. To perform TUNEL assay, transfected cells were fixed using 4% paraformaldehyde and then permeabilized using 0.25% Triton X-100, according to manufacturer's instructions. Cells were stained with a TUNEL assay (Click-iT TUNEL Alexa Fluor 594 Imaging Assay, Invitrogen, 10246) to identify those with fragmented DNA. Nuclei were counterstained with Hoechst 33342 (Life Technologies, CA, USA). Image acquisition was done using EVOS FLOID (Life Technologies) equipped with a 20 × Nikon objective.
5
Methanol Permeabilization of PBMCs
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PBMCs were isolated from the blood of healthy human donors at UNMC Elutriation Core facility. PBMCs were suspended in varying compositions of methanol (10 to 100% methanol solvent with water (vol/vol)). PBMCs and LNMCs collected from patients receiving ART were permeabilized in 70% methanol and were stored for more than one year at −80 °C. Following storage, we examined the morphology of cells under the microscope. Images of the cells were captured at 20X (fixed fluorite objective lens) magnification with a high sensitivity interline charge coupled device (CCD) monochrome camera fixed in EVOS FLOID (Life Technologies, Chicago, USA) cell imaging station. All images were processed and stored in JPEG file formats.
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