The FastFUCCI live-cell assay was performed as previously described (28 (link)). Briefly, cells were seeded in glass bottom chamber (ibidi GmbH) and were kept under cell culture conditions. Images were retrieved using a Nikon Eclipse TE2000-E microscope with a 20X long-working distance dry objective and a sCMOS Andor Neo camera. Red and green fluorescence were acquired using a pE-300white CoolLED source of light filtered by Nikon FITC B-2E/C and TRITC G-2E/C filter cubes, respectively. Live-cell time-lapse sequences were split into single channel sequences, and were applied with background subtraction and shading correction. Cell-tracking analysis was performed using the TrackMate plug-in available in the Fiji package.
Glass bottom chamber
Manufactured by Ibidi
Sourced in Germany
Ibidi glass-bottom chambers are laboratory equipment designed for cell culture experiments. They provide a transparent glass surface for cell attachment and observation. The chambers are available in various well configurations to accommodate different experimental setups.
Automatically generated - may contain errors
Lab products found in correlation
Iblot dry blotting system, by Thermo Fisher Scientific (2 mentions)
Sds page gel system, by Thermo Fisher Scientific (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Bio beads sm 2, by Bio-Rad (1 mentions)
5 protocols using glass bottom chamber
1
Live-cell imaging of cell cycle
2
Immunostaining and Immunoblotting Workflow
3
Munc13 Clustering on Lipid Membranes
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We adapted the protocol described previously (36 (link), 44 (link)) to assess the formation of Munc13 clusters on lipid membrane surface in the absence or presence of DAG/DHG. Liposomes were prepared with following lipid composition (71% DOPC, 25% DOPS, 2% PIP2, ±2% DAG) using extrusion method with HEPES buffer (50 mM HEPES, 140 mM KCl, 1 mM TCEP, pH 7.4). Lipid bilayers were created by Mg2+ (5 mM) induced bursting liposomes in ibidi glass-bottom chambers (ibidi GmbH, Germany) and extensively washed with the HEPES buffer supplemented with EDTA (6 mM). Munc13-Halo-Alexa488 (10 nM) was added to the prewashed bilayer and incubated for 60 mins. The samples were imaged on a TIRF (Nikon) microscope with a 63× oil objective. For DHG experiments, we added 500 nM DHG is solution along with Munc13.
4
Munc13 Clustering on Lipid Membranes
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We adapted the protocol described previously (36 (link), 44 (link)) to assess the formation of Munc13 clusters on lipid membrane surface in the absence or presence of DAG/DHG. Liposomes were prepared with following lipid composition (71% DOPC, 25% DOPS, 2% PIP2, ± 2% DAG) using extrusion method with HEPES buffer (50 mM HEPES, 140 mM KCl, 1 mM TCEP, pH 7.4). Lipid bilayers were created by Mg2+ (5 mM) induced bursting liposomes in ibidi glass-bottom chambers (ibidi GmbH, Germany) and extensively washed with the HEPES buffer supplemented with EDTA (6 mM). Munc13-Halo-Alexa488 (10 nM) was added to the pre-washed bilayer and incubated for 60 mins. The samples were imaged on a TIRF (Nikon) microscope with a 63x oil objective. For DHG experiments, we added 500 nM DHG is solution along with Munc13.
5
Stoichiometry of Syp-VAMP2 Complex
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To measure the stoichiometry of the Syp-VAMP2 complex on a supported bilayer, 1 mM (total) SUVs (60 (mole)% DOPC, 15% DOPS, 5% DOPE-PEG 2000 and 20% Cholesterol) were dried with N2 gas and kept under vacuum for another 2–4 hr. Dried lipid was dissolved with 25 mM HEPES, pH 7.4, 150 mM KCl, 0.2 mM TCEP and 2% Triton X-100. Pre-labeled and purified Syp-Halo (Alexa 488) and VAMP2 (Alexa 647) proteins were added into the mixture with a protein: lipid ratio of 1:20000 and 1:5000 respectively. 100 mg pre-washed Bio-beads SM2 (Bio-Rad, Hercules, CA) were incubated with that mixture for another 30 min at room temperature with gentle shaking. The liposomes were dialyzed overnight with a 6–8 kD cut-off against a detergent-free HEPES buffer. The liposomes were floated up with a discontinuous gradient of Opti prep. Samples were collected from the top carefully avoiding any Opti prep contamination and further dialyzed for another 2 hr. at 4°C. The supported bilayer was created by Mg2+ (5 mM) induced bursting of the liposomes in ibidi glass-bottom chambers (ibidi GmbH, Germany). The bilayer was extensively washed with HEPES buffer. The bilayer was imaged with TIRF (Nikon) microscope using 488 and 633 nm lasers. Photobleaching of the protein cluster was analyzed with ImageJ.
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