Palbociclib release from lysosomes was tested both on SK-Mel-103 and Saos2 cells. The cells were pre-treated with 1 µM palbociclib for 7 days and then seeded in a 6-channels flow chamber slides (IBIDI μSlide IV) in the presence of 4 μM palbociclib. The following day, a flux assay (200 μl/min) was performed simultaneously in two channels, injecting in one channel drug-free media and palbociclib (4 µM) in the other one. For the analysis of palbociclib uptake SK-Mel-103 were pre-treated with 1 µM palbociclib for 7 days and then seeded in a 6-channels flow chamber slides (IBIDI μSlide IV) in the absence of palbociclib. The following day, media containing palbociclib (4 µM) was continuously fluxed through the cultures (200 μl/min). Pictures were taken every 10 minutes with a DMI6000B microscope (Leica microsystems): 405 nm excitation and detection with a 500–550 nm filter. The fluorescence signal of the cells and the background in each frame was quantified with ImageJ. At least 10 individual cells were analyzed in each experimental condition.
μ slide 4
Manufactured by Ibidi
Sourced in Germany
The μ-Slide IV is a specialized lab equipment designed for various cell-based applications. It provides a four-channel flow chamber system for live-cell imaging and other experiments. The μ-Slide IV facilitates controlled fluid delivery and enables simultaneous observation of multiple samples under a microscope.
Automatically generated - may contain errors
4 protocols using μ slide 4
1
Palbociclib Release from Lysosomes
2
Neutrophil NETosis and Microparticle Interactions
3
Immunofluorescence Imaging of γ-Synuclein
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RGC-5 cells were grown in μ-slide IV (Ibidi GmbH, München, Germany) over night and subsequently washed with PBS. Then the cells were fixed with 3% paraformaldehyde (15 min), incubated with 0.25% Triton-X-100 in PBS (12 min), washed 3× with PBS and treated with 1% BSA (20 min). After incubating the cells with 2 µg/ml sheep polyclonal anti-γ-synuclein abs over-night they were gently washed 3× with PBS and incubated with rabbit anti sheep IgG-H&L conjugated with FITC for 1.5 h. They were visualized with Leica fluorescence microscope using Lucia G/F software after washing them with PBS. To investigate the ab uptake in living cells the cells were incubated with 15 µg/ml sheep polyclonal anti-γ-synuclein abs and washed with PBS. The cell membrane was visualized using wheat germ agglutinin.
4
Visualizing 14-3-3 sigma in RGC-5 cells
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RGC-5 cells were grown in μ-slide IV (Ibidi GmbH, Munich, Germany) and subsequently washed with PBS. Then the cells were fixed with 3 % paraformaldehyde for 15 min and incubated with 0.25 % Triton-X-100 for 12 min. After 3 wash steps with PBS, the cells were treated with 1 % bovine serum albumin for 20 min. Afterwards, the cells were incubated with 2 μg/ml rabbit polyclonal anti 14-3-3 sigma antibodies overnight, then gently washed 3 times with PBS and incubated with Goat polyclonal secondary antibody to rabbit IgG-H&L conjugated with FITC for 1.5 h. After 3 washing steps with PBS the cells were visualized with a fluorescence microscope (Leica Microsystem, Heidelberg, Germany). To investigate the antibody uptake in living cells, the cells were preincubated with 10 μg/ml rabbit polyclonal anti 14-3-3 sigma abs for 3 h and then washed with PBS to remove unbound antibodies. Controls were preincubated with medium not containing the polyclonal anti 14-3-3 sigma abs. The cells then were treated as described above and visualized with a Leica fluorescence microscope and using Lucia G/F software.
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