To embed organoids in collagen droplets, a sheet of Parafilm was soaked in 70 % ethanol, air dried, and pressed against the holes of a box of gel-loading pipette tips (1-200 μl, Fisher Scientific 02-707-138) to create a dimpled mold31 (link). One organoid was placed in each dimple and 30 μL of 7 mg/mL collagen I (Corning) was added. The droplets were incubated 25 minutes at 37°C and carefully resuspended in 3 ml of RB media in an untreated 6-well plate. The media was changed weekly and the droplets were imaged after 2 weeks of incubation using a Nikon Ti Inverted Widefield microscope and a Nikon 1 J1 Camera. Droplet diameters were measured using NIS Elements imaging software (Nikon) and normalized to the diameter of empty droplets from the same set, for a total of three sets. Droplets that failed to undergo compaction (∼ 20 % in control and ∼ 50 % in PKD samples) were excluded. Droplets were fixed with 4% paraformaldehyde for 20 minutes at room temperature, incubated 16 hours in 30 % sucrose (Sigma) in water, mounted in Tissue-Tek (Sakura), flash frozen, and cryosectioned onto SuperfrostPlus slides (Fisher). Sections were stained in Picro-sirius red solution (Sky-Tek laboratories) for one hour, rinsed in two changes of 0.5% acetic acid solution, and dehydrated in two changes of absolute ethanol before mounting. Immunofluorescence was performed as described below.
Gel loading pipette tips
Manufactured by Thermo Fisher Scientific
Gel-loading pipette tips are specialized laboratory equipment designed to facilitate the accurate and precise transfer of small liquid volumes, particularly for use in gel electrophoresis applications. These tips are engineered with a narrow, elongated shape to allow for easy and controlled delivery of samples into gel wells.
Automatically generated - may contain errors
Lab products found in correlation
Nis elements imaging software, by Nikon (2 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (2 mentions)
Collagen 1, by Corning (2 mentions)
Ti inverted widefield microscope, by Nikon (2 mentions)
1 j1 camera, by Nikon (2 mentions)
Digitizer through pclamp 10, by Molecular Devices (2 mentions)
4 protocols using gel loading pipette tips
1
Embedding Organoids in Collagen Droplets
2
Whole-cell patch-clamp recordings and NO donor injection
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Whole-cell patch-clamp recordings were performed with MultiClamp 700B amplifier and Digidata 1440A digitizer (Axon Instrument) at room temperature. The micropipettes were fabricated from borosilicate glass tubes (O.D.: 1.5 mm, I.D.: 0.86 mm, BF150−86−7.5, Shutter Instrument) with a flaming brown micropipette puller (P−80/PC, Shutter Instrument). The intracellular solution was comprised of 142 mM K-gluconate, 2 mM KCl, 0.2 mM ethylene glycol tetraacetic acid (EGTA), 4 mM MgATP, 10 mM HEPES, 7 mM Na2-phosphocreatine. The pH was adjusted to 7.4. The extracellular solution was comprised of 125 mM NaCl, 2 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 25 mM HEPES, and 51 mM D-glucose. The pH was adjusted to 7.4. Cells were perfused with a gravity-fed perfusion system at a rate of 1−2 mL/min before stimulation. Pulse sequences were formed by a Digitizer through pClamp 10.2 (Molecular Devices). Signals were sampled at 10 kHz and were then low-pass filtered at 1 kHz. The total membrane capacitance was estimated from the surface area of the cell, according to the previous report47 . Due to its short lifetime, NO donor was rapidly injected into the extracellular solution with gel-loading pipette tips (Fisher Scientific).
3
Embedding Organoids in Collagen Droplets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To embed organoids in collagen droplets, a sheet of Parafilm was soaked in 70 % ethanol, air dried, and pressed against the holes of a box of gel-loading pipette tips (1-200 μl, Fisher Scientific 02-707-138) to create a dimpled mold31 (link). One organoid was placed in each dimple and 30 μL of 7 mg/mL collagen I (Corning) was added. The droplets were incubated 25 minutes at 37°C and carefully resuspended in 3 ml of RB media in an untreated 6-well plate. The media was changed weekly and the droplets were imaged after 2 weeks of incubation using a Nikon Ti Inverted Widefield microscope and a Nikon 1 J1 Camera. Droplet diameters were measured using NIS Elements imaging software (Nikon) and normalized to the diameter of empty droplets from the same set, for a total of three sets. Droplets that failed to undergo compaction (∼ 20 % in control and ∼ 50 % in PKD samples) were excluded. Droplets were fixed with 4% paraformaldehyde for 20 minutes at room temperature, incubated 16 hours in 30 % sucrose (Sigma) in water, mounted in Tissue-Tek (Sakura), flash frozen, and cryosectioned onto SuperfrostPlus slides (Fisher). Sections were stained in Picro-sirius red solution (Sky-Tek laboratories) for one hour, rinsed in two changes of 0.5% acetic acid solution, and dehydrated in two changes of absolute ethanol before mounting. Immunofluorescence was performed as described below.
4
Whole-cell patch-clamp recordings and NO donor injection
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