Border cell migration was observed in egg chambers expressing rk RNAi (VDRC, line v29931) driven by slbo-Gal4. Cell membranes were visualized with a Gap43-mCherry marker (Martin et al., 2010 (link)), which labels the membranes of all cells, with especially strong signal surrounding the polar cells. Females were fed yeast for 40 h prior to dissection. Individual ovarioles were dissected out of the ovary, and the germaria and older egg chambers were removed from these ovarioles. Dissection was performed in media previously described (Prasad et al., 2007 (link); Domanitskaya et al., 2014 (link)). Droplets of media containing dissected ovarioles were imaged with a Nikon Ti-E spinning disc confocal microscope equipped with a Perfect Focus System, a Yokogawa spinning disc, and a Hamamatsu detector. We imaged border cell cluster detachment and early migration for approximately 4–6 h in rk RNAi expressing egg chambers, and wild type egg chambers expressing the Gap43-mCherry membrane marker as a control. A 561 nm laser was utilized to detect the mCherry signal. 10–20 μm Z stacks were imaged to capture the whole border cell cluster, with Z stacks containing 1 μm steps taken every 3 min.
Spinning disc
Manufactured by Yokogawa
The Spinning Disc is a laboratory equipment that utilizes a rotating disc to perform various experimental applications. The device consists of a flat, circular platform that spins at a controlled speed, allowing for the study of processes affected by centrifugal force.
Automatically generated - may contain errors
Lab products found in correlation
Alpha plan apochromat 100x objective, by Zeiss (2 mentions)
Orca r2 camera, by Hamamatsu Photonics (2 mentions)
Ultraview vox, by PerkinElmer (2 mentions)
Bx41 fluorescence microscope, by Yokogawa (2 mentions)
Coolsnaphq2 camera, by Universal Imaging (2 mentions)
Hc pl apo cs2 93 x 1.30 glyc objective, by Leica camera (2 mentions)
Sp8 confocal microscope, by Leica camera (2 mentions)
Metaview software, by Universal Imaging (2 mentions)
Mcl 400, by Agilent Technologies (2 mentions)
Eclipse ti inverted microscope, by Nikon (2 mentions)
Perfect focus system, by Yokogawa (1 mentions)
Tie spinning disc confocal microscope, by Yokogawa (1 mentions)
Du 888, by Oxford Instruments (1 mentions)
Attofluor cell chamber, by Thermo Fisher Scientific (1 mentions)
Genjet, by SignaGen (1 mentions)
T25 flask, by Corning (1 mentions)
Back illuminated emccd camera, by Oxford Instruments (1 mentions)
7 protocols using spinning disc
1
Imaging border cell migration in Drosophila
2
Visualizing ASC Dynamics in Yeast Cells
3
Visualizing ASC Dynamics in Yeast Cells
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Yeast cells expressing ASC-mEos3.1 were grown overnight in synthetic media containing 2% dextrose while shaken at 30 °C. Cells were then loaded into a CellASIC ONIX microfluidic device (Millipore Sigma B04A03). Media containing 2% galactose was flowed through the microfluidic device at a rate of 5 kPa from 2 wells at a time. Timelapse images were acquired on an Ultraview Vox (Perkin Elmer) Spinning Disc (Yokogawa CSU-X1). Images were collected with an alpha Plan Apochromat 100x objective (Zeiss, NA 1.4) onto an Orca R2 camera (Hamamatsu, C10600–10B). mEos3.1 was excited with a 488 nm laser, and the fluorescence emission was collected through a 525–50 nm bandpass filter. Images were collected as z-stacks with 0.5 lm steps (41 slices) and a 30 ms camera integration time every 5 minutes. Additionally, a single transmitted light image was acquired in the middle of the z stack with an integration time of 200 ms. Each time point was sum projected and the resulting time course was registered to reduce movement of the cells. Regions of interest were then drawn in individual cells and the mean intensity of the sum projected fluorescence was measured from the beginning of the time course until the end.
4
Immunostaining and Confocal Microscopy Protocol
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Immunostaining was observed with an Olympus BX41 fluorescence microscope (objective 40 X/1.30 or 63 X/1.25) equipped with a Yokogawa spinning disc and a CoolSnapHQ2 camera driven by Metaview software (Universal Imaging).
For co-immunodetection of Scabrous and Delta, images were acquired with a Leica SP8 confocal microscope, with the HC PL APO CS2 93 X/1.30 GLYC objective. We tuned the white light laser (WLL) to 650 nm for the excitation. The detector was a HyD. The pixel size was 0.087 µm and the z step size was 0.332 µm. Deconvolution was done with Huygens software. All images were processed with Fiji software (Schindelin et al., 2012 (link)).
For co-immunodetection of Scabrous and Delta, images were acquired with a Leica SP8 confocal microscope, with the HC PL APO CS2 93 X/1.30 GLYC objective. We tuned the white light laser (WLL) to 650 nm for the excitation. The detector was a HyD. The pixel size was 0.087 µm and the z step size was 0.332 µm. Deconvolution was done with Huygens software. All images were processed with Fiji software (Schindelin et al., 2012 (link)).
5
Multimodal Microscopy for Protein Localization
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Immunostaining was observed with an Olympus BX41 fluorescence microscope (objective 40X/1.30 or 63X/1.25) equipped with a Yokogawa spinning disc and a CoolSnapHQ2 camera driven by Metaview software (Universal Imaging).
For co-immunodetection of Scabrous and Delta, images were acquired with a Leica SP8 confocal microscope, with the HC PL APO CS2 93 X/1.30 GLYC objective. We tuned the white light laser (WLL)
to 650 nm for the excitation. The detector was a HyD. The pixel size was 0.087 µm and the z step size was 0.332 µm. Deconvolution was done with Huygens software. All images were processed with Fiji software 41 .
For co-immunodetection of Scabrous and Delta, images were acquired with a Leica SP8 confocal microscope, with the HC PL APO CS2 93 X/1.30 GLYC objective. We tuned the white light laser (WLL)
to 650 nm for the excitation. The detector was a HyD. The pixel size was 0.087 µm and the z step size was 0.332 µm. Deconvolution was done with Huygens software. All images were processed with Fiji software 41 .
6
Live-cell Imaging of EB3 Comets
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Cells were plated on coverslips and transfected with a plasmid encoding mCherry-EB3 (a kind gift from Dr. Irina Kaverina, Vanderbilt University, Nashville, TN) using GenJet (SignaGen Laboratories) following the manufacturer’s instructions. ∼24 h after transfection, cells were mounted in Attofluor Cell chambers (A7816; Thermo Fisher Scientific) and imaged using an Eclipse Ti inverted microscope (Nikon) equipped with a Yokogawa spinning disc (Yokogawa Electric Corp.), 405-, 488-, 561-, and 640-nm laser launch (MCL-400; Agilent Technology), and a back-illuminated EMCCD camera (DU888; Andor), 100×, NA 1.42, Plan Apo objective, with a 2× relay lens placed before the spinning disc. A time-lapse over 1 min was recorded imaging one z plane each 1 s, using 200-ms exposure time. 4-s time projections were generated in Fiji (National Institutes of Health). The length of the comet over a 4-s period was determined if the same comet appeared over more than one 4-s timeframes. The displacement of the EB3 comets in 1 s was calculated and plotted.
7
Validating PSMD2 Biotin Integration
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After 6 weeks of incubation post FACS-sorting, six clones from the 96-well plate exhibited cell growth. These clones were trypsinized and expanded into a T25 flask (Corning; 430639) and incubated at 37 °C with 5% CO2 humidity for an additional 2 weeks of growth. To validate the integration of the biotin tag into PSMD2, live cells expressing mScarlet were visualized by confocal microscopy. Imaging was performed by an Eclipse Ti inverted microscope (Nikon Inc) equipped with a Yokogawa spinning disc (Yokogawa Electric Corporation), back-illuminated EMCCD camera (Andor, DU888), and a 2x relay lens placed before the spinning disc. A 60x, NA 1.45, Plan Apo objective and a 561 nm laser (Agilent Technology MCL-400) was used to acquire fluorescence (mScarlet) images in parallel with differential interference contrast images for reference.
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