HEK293 cells were used to generate stable transfections of wild type and proline mutants of αIIbβ3. The single cell clones that have comparable surface expression of αIIbβ3 were selected for cell adhesion and spreading assay as described before (38 (link), 40 (link)). In brief, the Delta T dish (Bioptechs) was coated with 5 μg/ml PAC-1 or 25 μg/ml fibrinogen in PBS buffer at 4 °C overnight and then blocked with 1% BSA at 37 °C for 1 h. The cells in suspension were washed once with DMEM without serum and seeded onto the Delta T dish at 37 °C for 1 h. The attached cells were washed with DMEM and fixed with 3.7% formaldehyde in PBS at 25 °C for 5 min. The fixed cells were first immunostained with mAb AP3 and then permeabilized with 0.05% Triton X-100 in PBS, followed by staining with Alexa Fluor 546 labeled phalloidin (Thermo Fisher Scientific) and DAPI. Cells were imaged with EVOS digital inverted fluorescence microscope. The cell areas of 40 to 50 total cells for each independent experiment were measured using ImageJ and averaged.
Delta t dish
Manufactured by Bioptechs
Sourced in United States
The Delta-T dish is a specialized petri dish designed for live cell imaging. It maintains the optimal temperature and environment for cells during microscopic observation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Perfusable coverglass lid, by Bioptechs (2 mentions)
Virapower packaging system, by Thermo Fisher Scientific (1 mentions)
X tremegene hp, by Roche (1 mentions)
Halogen lamp light source, by Sutter Instruments (1 mentions)
Matlab, by MathWorks (1 mentions)
Fluo 4, by Thermo Fisher Scientific (1 mentions)
Ix81 inverted microscope, by Olympus (1 mentions)
Alexafluor 546 labeled phalloidin, by Thermo Fisher Scientific (1 mentions)
7 protocols using delta t dish
1
Characterizing Cell Adhesion and Spreading
2
Live-cell and PALM imaging of dimerized proteins
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U2OS cells (HTB-96, ATCC) were cultured at 37°C and 5% CO2 in DMEM supplemented with 10% FBS (11995 and 10082 respectively, Life Technologies). Cells were plated in phenol red-free DMEM (21063, Life Technologies) supplemented with 10% FBS on a #1.5 Lab-Tek chamber slide (155409, Thermo Scientific) for PALM imaging after fixation, or a 0.17 mm coverslip bottom Delta T Dish (04200417, Bioptechs) for live cell imaging. Plasmids for the artificial dimerization system were transiently transfected using X-tremeGENE HP (13873800, Roche) as described by the manufacturer. Dimerization was induced by adding 500 nM A/C Heterodimerizer (635057, Clontech) and incubating at 37°C for 2 hours or overnight as indicated. KRas G12D and CRaf RBD constructs were introduced into the cells by lentiviral infection using the ViraPower packaging system (K497500, Life Technologies). For PALM imaging, cells were fixed in fresh 3.7% PFA with 0.1% glutaraldehyde for 15 minutes at room temperature and changed to imaging buffer (100 mM Tris with 30 mM NaCl and 20 mM MgCl2, pH 8.5) after fixation. Gold particles (100 nm, EM.GC100, BBI International) were added as fiducial markers to correct for stage drift during imaging.
3
Calcium Flux Imaging of C57 Cells
4
Monitoring Lipid Signaling in SMCs
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Rat aortic SMCs were transfected with CFP-GRAF3-BAR-PH-GAP-YFP plasmid. The next day, cells were plated on a delta T dish (Bioptechs, Butler, PA, USA) and starved for 4 h prior to treatment with 10 μM Sphingosine 1 phosphate (S1P). CFP and FRET signals were excited at 440 nm and emission signals were collected at 470 nm and 535 nm, respectively, using an Olympus IX-81 inverted microscope. Image J software was used to calculate FRET signals. The FRET/CFP ratio of each cell was normalized to the FRET/CFP value before S1P stimulation.
5
Imaging Live Avian Embryos Ex Ovo
6
Visualizing Collagen Fibril Dynamics
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A glass bottom, ITO coated Delta-T dish (04200417C, Bioptechs) and a perfusable coverglass lid (0420031216, Bioptechs) were plasma cleaned and coated with 1% BSA at 4 °C overnight. The dish was placed on the microscope stage and 1 mL of sclera fibril suspension was added. After an hour incubation for fibril attachment, 20 mL of 1X PBS was perfused into the dish at 20 mL/h using a syringe pump. A temperature controller (5410429, Bioptechs) was set at either 25 or 30 °C, and the fibrils were imaged prior to the addition of labeled monomers. Next 400 µL of 10 µg/mL AF488-Col (DOL of ~0.5) was added to the dish at 12 mL/h for 2 minutes to rapidly adjust the collagen concentration inside the dish to 2 µg/mL (perfect mixing was assumed). Then 5 mL of 2 µg/mL AF488-Col was added at 1 mL/h to maintain constant concentration inside the chamber.
The collagen concentration was kept at 2 µg/mL (sub-threshold for new fibril formation) to prevent self-assembly [27c, 49] and formation of new fibrils independent of the native fibril in the dish. DIC and fluorescent images were taken every 10 minutes for 3 hours. As a control experiment, AF488-Col was added to the dish in the absence of native scleral fibrils to ensure that collagen concentration was at sub-threshold for new fibril formation.
The collagen concentration was kept at 2 µg/mL (sub-threshold for new fibril formation) to prevent self-assembly [27c, 49] and formation of new fibrils independent of the native fibril in the dish. DIC and fluorescent images were taken every 10 minutes for 3 hours. As a control experiment, AF488-Col was added to the dish in the absence of native scleral fibrils to ensure that collagen concentration was at sub-threshold for new fibril formation.
7
Visualizing Collagen Fibril Dynamics
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A glass bottom, ITO coated Delta-T dish (04200417C, Bioptechs) and a perfusable coverglass lid (0420031216, Bioptechs) were plasma cleaned and coated with 1% BSA at 4 °C overnight. The dish was placed on the microscope stage and 1 mL of sclera fibril suspension was added. After an hour incubation for fibril attachment, 20 mL of 1X PBS was perfused into the dish at 20 mL/h using a syringe pump. A temperature controller (5410429, Bioptechs) was set at either 25 or 30 °C, and the fibrils were imaged prior to the addition of labeled monomers. Next 400 µL of 10 µg/mL AF488-Col (DOL of ~0.5) was added to the dish at 12 mL/h for 2 minutes to rapidly adjust the collagen concentration inside the dish to 2 µg/mL (perfect mixing was assumed). Then 5 mL of 2 µg/mL AF488-Col was added at 1 mL/h to maintain constant concentration inside the chamber.
The collagen concentration was kept at 2 µg/mL (sub-threshold for new fibril formation) to prevent self-assembly [27c, 49] and formation of new fibrils independent of the native fibril in the dish. DIC and fluorescent images were taken every 10 minutes for 3 hours. As a control experiment, AF488-Col was added to the dish in the absence of native scleral fibrils to ensure that collagen concentration was at sub-threshold for new fibril formation.
The collagen concentration was kept at 2 µg/mL (sub-threshold for new fibril formation) to prevent self-assembly [27c, 49] and formation of new fibrils independent of the native fibril in the dish. DIC and fluorescent images were taken every 10 minutes for 3 hours. As a control experiment, AF488-Col was added to the dish in the absence of native scleral fibrils to ensure that collagen concentration was at sub-threshold for new fibril formation.
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