HeLa pLuc/705 7 × 103 cells were seeded in a 96-well plate with glass bottom 24 h before treatment. Cells were incubated in serum-containing media with/without ligand for 1 h; then the fluorescent PF14/SCO(Alexa568) complexes (MR10, 100 nM SCO) were applied and incubated with cells for 24 h. Cells were then stained with Cyto-ID kit (Enzo Life Sciences) according to the manufacturer’s instruction. Imaging was performed using Leica DM/IRBE 2 epi-fluorescence microscope controlled by Micro-Manager19 with a 63 × 1.4 NA oil immersion objective. Images were analyzed using Fiji (ImageJ) software20 . Quantification was performed by acquiring a stack with 0.3 µm slices through the cell. The stack was filtered with the 3D mean filter using 2 × 2 × 2 setting. Cells were then manually marked as a region of interest (ROI) and the signal from the ROIs with the highest average intensity used. Cell-free ROIs next to the measured cell was used for background subtraction.
Dm irbe 2 epi fluorescence microscope
Manufactured by Leica
The DM/IRBE 2 epi-fluorescence microscope is a high-performance microscope designed for a wide range of applications. It features an epi-fluorescence illumination system, allowing for the observation of fluorescently labeled samples. The microscope is equipped with a variety of objectives and filter sets, enabling versatile imaging capabilities.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using dm irbe 2 epi fluorescence microscope
1
Quantification of Cellular Uptake of SCO-Complexes
2
Epifluorescence Microscopy Imaging of CPPs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To establish the peptides function as CPPs, epi-fluorescence microscopy imaging was used. 15k cells were seeded in each well of a microscopy glass bottom black 96-well plate, left attaching overnight and then treated with various concentration of the fluorophore-conjugated peptide (ranging from 10nM to 50µM) at different points in time (1hr, 3hrs, 24hrs), followed by 15 min incubation with the nuclear stain (Hoechst 33342). To remove excess stain from the plate, the wells were then rinsed twice using Opti-MEM Reduced Serum Medium. To ensure cell viability, 200µL of the medium were added to each well before the microscopy.
Imaging was performed using a Leica DM/IRBE 2 epi-fluorescence microscope with 10X, 43X dry or 43X, 63 × 1.4 NA oil immersion objective. Samples were kept at 37°C at all times. Images were recorded via a Hamamatsu Orca-ER CCD camera. The system was controlled by Micro-Manager (Edelstein et al. 2014) (link). Images were then processed using Image J (Rueden et al. 2017) (link).
Imaging was performed using a Leica DM/IRBE 2 epi-fluorescence microscope with 10X, 43X dry or 43X, 63 × 1.4 NA oil immersion objective. Samples were kept at 37°C at all times. Images were recorded via a Hamamatsu Orca-ER CCD camera. The system was controlled by Micro-Manager (Edelstein et al. 2014) (link). Images were then processed using Image J (Rueden et al. 2017) (link).
3
Peptide Cellular Uptake Imaging Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To establish the peptides function as CPPs, epi-fluorescence microscopy imaging was used. 15 k cells were seeded in each well of a microscopy glass bottom black 96-well plate, left attaching overnight and then treated with various concentration of the fluorophore-conjugated peptide (ranging from 10 nM to 50 µM) at different points in time (1 h, 3 h, 24 h), followed by 15 min incubation with the nuclear stain (Hoechst 33342). To remove excess stain from the plate, the wells were then rinsed twice using Opti-MEM Reduced Serum Medium. To ensure cell viability, 200 µL of the medium were added to each well before the microscopy.
Imaging was performed using a Leica DM/IRBE 2 epi-fluorescence microscope with 10 ×, 43 × dry or 43 ×, 63 × 1.4 NA oil immersion objective. Samples were kept at 37 °C at all times. Images were recorded via a Hamamatsu Orca-ER CCD camera. The system was controlled by Micro-Manager (Edelstein et al. 2014) (link). Images were then processed using Image J (Rueden et al. 2017) (link).
Imaging was performed using a Leica DM/IRBE 2 epi-fluorescence microscope with 10 ×, 43 × dry or 43 ×, 63 × 1.4 NA oil immersion objective. Samples were kept at 37 °C at all times. Images were recorded via a Hamamatsu Orca-ER CCD camera. The system was controlled by Micro-Manager (Edelstein et al. 2014) (link). Images were then processed using Image J (Rueden et al. 2017) (link).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!