For single-cell gliding on glass, cells were grown in CYE at 28°C to an A600 nm ≈ 0.7. Cells were diluted to an A600 nm ≈ 0.05 and 100 μL were spotted into μ-Slide chambers with glass coverslip bottom (Ibidi). After 5-minute incubation, floating cells were washed out with fresh CYE medium and gliding of adherent cells was monitored by phase contrast microscopy on a Nikon Eclipse TE-2000 microscope equipped with a 100× NA 1.3 Ph3 objective, a perfect focus system to maintain the plane in focus, and an Orcaflash 4.0 LT digital camera (Hamamatsu Photonics, Shizuoka, Japan). GldL-alfa/NBalfa-sfGFP localization was observed by Hilo microscopy. Cells were grown in CYE overnight without shaking at 28°C. NBalfa-sfGFP expression was induced with 1 mM IPTG for 1 hour prior to observation. Cells were spotted on a 2% low-melting agarose pad for immediate observation. Hilo fluorescence microscopy and FRAP experiments were performed with a Nikon Eclipse Ti2 microscope equipped with a 100x NA 1.45 Ph3 objective, an Orca-Fusion digital camera (Hamamatsu Photonics), a perfect focus system, and an Ilas2 TIRF/FRAP module (Gataca Systems, Massy, France).
Glass coverslip bottom
Manufactured by Ibidi
Sourced in Germany
The glass coverslip bottom is a transparent substrate used in various microscopy applications. It provides a flat, smooth surface for sample mounting and imaging. The product is made of high-quality glass, ensuring optical clarity and durability.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse te2000 microscope, by Nikon (1 mentions)
μ slide chamber, by Ibidi (1 mentions)
Nikoneclipse ti2 microscope, by Hamamatsu Photonics (1 mentions)
Orca flash 4.0 lt digital camera, by Hamamatsu Photonics (1 mentions)
Orca fusion digital camera, by Hamamatsu Photonics (1 mentions)
L 15 medium, by Merck Group (1 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Snap cell 647 sir, by New England Biolabs (1 mentions)
3 protocols using glass coverslip bottom
1
Single-cell Gliding Motility Assay
2
Fluorescent Tagging of Human Fibroblasts
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Cells were prepared using the same protocol as
in ref (19 (link)). Human
fibroblasts (GM5756T, Moser, Baltimore, U.S.A.) were maintained in
a culture medium consisting of DMEM with 4500 mg glucose/L, 110 mg
sodium pyruvate/L supplemented with 10% fetal calf serum,l -glutamine (2 mM), and penicillin–streptomycin (1%). The cells
were cultured at 37 °C/5% CO2. Cells were grown in
a 35 mm imaging dish with a glass coverslip bottom (ibidi GmbH, Germany)
and transfected with 2.5 μg DNA per dish of a plasmid expressing
a fusion protein of GFP and SNAP-tag using Lipofectamine 3000 transfection
reagent (Invitrogene, Carlsbad, U.S.A.). A total of 24 h after transfection,
the cells were incubated together with SNAP-Cell 647-SiR (New England
Biolabs (U.K.) Ltd., Hitchin, U.K.) for 40 min and washed twice with
culture medium, with a waiting time between washings of 20 min. The
culture medium was finally substituted with L-15 medium (Sigma-Aldrich,
Dorset, U.K.), and each sample was visualized at 37 °C for no
longer than 1 h.
in ref (19 (link)). Human
fibroblasts (GM5756T, Moser, Baltimore, U.S.A.) were maintained in
a culture medium consisting of DMEM with 4500 mg glucose/L, 110 mg
sodium pyruvate/L supplemented with 10% fetal calf serum,
were cultured at 37 °C/5% CO2. Cells were grown in
a 35 mm imaging dish with a glass coverslip bottom (ibidi GmbH, Germany)
and transfected with 2.5 μg DNA per dish of a plasmid expressing
a fusion protein of GFP and SNAP-tag using Lipofectamine 3000 transfection
reagent (Invitrogene, Carlsbad, U.S.A.). A total of 24 h after transfection,
the cells were incubated together with SNAP-Cell 647-SiR (New England
Biolabs (U.K.) Ltd., Hitchin, U.K.) for 40 min and washed twice with
culture medium, with a waiting time between washings of 20 min. The
culture medium was finally substituted with L-15 medium (Sigma-Aldrich,
Dorset, U.K.), and each sample was visualized at 37 °C for no
longer than 1 h.
3
Detecting Intracellular Lipid Aggregates in MEF Cells
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For detecting intra-cellular lipid aggregates, 70 % confluent MEF cell culture was supplemented with 1 μM DOXSA from ethanolic stock or vehicle (0.1 % ethanol). After incubating for 24 h, cells were treated with HCS LipidTOX Red Phospholipidosis detection Reagent (LipidTOX red) (Life Technologies, Darmstadt, Germany) according to manufacturer's instructions. After aldehyde fixation cells were immediately imaged using LSM710 NLO in in µ-dish 35 mm imaging dishes with a glass cover slip bottom (Ibidi, Munich, Germany).
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