Locust primary brain cell cultures were established from 4th stage juvenile locusts as previously described (Ostrowski et al., 2011 (link); Miljus et al., 2014 (link)). Complete growth medium consisted of L15 (Leibovitz’s L-15 Medium, #11415049, Thermo Fisher Scientific, Germany), 5% FBSG (Fetal Bovine Serum Gold, PAA Laboratories GmbH, Austria), 1x Penicillin-Streptomycin (Penicillin-Streptomycin, 10,000 units penicillin and 10 mg streptomycin/ml, #P4333, Sigma-Aldrich®, Germany) and 1% Amphotericin B (GibcoTM Amphotericin B, 250 μg/ml, #15290018, Thermo Fisher Scientific, Germany). Dissected brains were pooled (see below), enzymatically digested with 2 mg/ml Collagenase/Dispase solution for 30–45 min at 27°C and mechanically dissociated by trituration with a 100 μl tip of an Eppendorf pipette. The primary brain cells were cultured on ConcanavalinA-coated (Sigma-Aldrich®, Germany) round glass cover slips (Ø 10mm, Corning, Inc., Sigma-Aldrich®, Germany) in 4-well NUNC plates (#176740, NuncTM Delta Surface, Thermo Fisher Scientific, Germany) filled with 500 μl of complete growth medium at 27°C in a humidified atmosphere. The medium was changed every 2 days. Based on previous studies (Gocht et al., 2009 (link)), locust brain cultures are estimated to contain approximately 3% glia and 97% neurons after 7 days in vitro under normoxic conditions.
Glass coverslip
Manufactured by Corning
Sourced in United States
Glass coverslips are thin, flat pieces of glass used as a covering or support for microscopic specimens. They serve as a protective barrier, allowing for clear observation and analysis of the samples.
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Lab products found in correlation
Alexa fluor 555, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole dihydrochloride, by Thermo Fisher Scientific (2 mentions)
Dmi4000 inverted fluorescent microscope, by Leica (2 mentions)
Bluemask 1, by ChemoMetec (2 mentions)
Xcyto 2 sample slides, by ChemoMetec (2 mentions)
Cellinsight cx7, by Thermo Fisher Scientific (2 mentions)
L 15 medium, by Merck Group (2 mentions)
Evos fl imaging system, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 555 conjugated anti human igg antibody, by Thermo Fisher Scientific (2 mentions)
Af555 conjugated anti rabbit igg antibody, by Thermo Fisher Scientific (2 mentions)
Concanavalin a coated, by Merck Group (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Fetal bovine serum gold, by GE Healthcare (1 mentions)
Axio imager z1, by Zeiss (1 mentions)
Evos fl auto microscope, by Thermo Fisher Scientific (1 mentions)
Duolink pla rabbit minus, by Merck Group (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Cox 4, by Cell Signaling Technology (1 mentions)
Ultraview ers, by Zeiss (1 mentions)
Cyclin b1 gns1, by SCBT (1 mentions)
32 protocols using glass coverslip
1
Locust Primary Brain Cell Culture
2
Microscope Slide and Coverslip Preparation
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We purchased glass coverslips (Corning, 22
mm × 22 mm) and premium plain glass microscope slides (75 mm
× 25 mm) from Thermo Fisher Scientific (Waltham, MA).
mm × 22 mm) and premium plain glass microscope slides (75 mm
× 25 mm) from Thermo Fisher Scientific (Waltham, MA).
3
Immunofluorescence Staining of Fibroblasts
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Fibroblasts were plated in 6-well plates containing glass coverslips (Corning, New York, NY, USA) at a cell density of 75,000 cells/well for 24 h. Then, cells were fixed using 4% paraformaldehyde for 20 min and permeabilized with 0.2% Triton X-100/1× PBS for 10 min. After blocking with 3% BSA/1× PBS for 1 h, the cells were incubated with specific primary antibodies (Table 1 ) in 3% BSA/1× PBS for 2 h at room temperature, followed by incubation with the appropriate secondary antibody (Table 1 ) in 3% BSA/1× PBS for 1 h in the dark. The coverslips were mounted on a microscope slide using Vectashield® mounting medium with 4′,6-diamidino-2-phenolyde (DAPI) (Vector Laboratories, Burlingame, CA, USA) [27 (link),74 (link)]. Image acquisition was performed using an epifluorescence microscopy Zeiss AxioImager Z1 (Zeiss, Jena, Germany) motorized microscope equipped with a Plan-ApoCHROMAT 63×/1.4 oil objective lens. Microphotograph images were taken with a digital AxioCam HR3 (soft imaging system).
4
Fabrication of Microfluidic Devices for Microalgae
5
Proximity Ligation Assay for MUL1-AKT Interaction
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SCC15 or SCC-QLL1 human HNC cells were attached on glass cover slips (Corning, Lowell, MA) at 70% confluence and cultured overnight. On the next day, cells were washed with PBS, treated with NTP, and cultured for an additional 24 hours in the absence of serum. The cells were fixed in 4% paraformaldehyde for 15 min, permeablized with 0.1% Triton X-100, and incubated with 5% BSA for 1 hour at room temperature. Cells were incubated with rabbit anti-MUL1 (1:200) and mouse anti-pan-AKT (1:200) antibodies at 4°C overnight. After washing, the slides were incubated with Duolink PLA Rabbit MINUS and PLA Mouse PLUS proximity probes, and proximity ligation was performed using the Duolink® with PLA® Technology reagent (Sigma, St. Louis, MO) according to the manufacturer's protocol. Fluorescence was detected using an EVOS FL AUTO microscope (Life Technology, NY, USA).
6
Visualizing Microtubule Dynamics in MCF-7 Cells
7
Polymer Film Deposition on Glass Coverslips
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Films of polymer
on glass coverslips (Corning, 22 mm × 22 mm) were prepared by
applying 300 μL of 1 wt % (w/w) polymer on a coverslip and evenly
spreading the solution with the side of a pipette tip.66 (link) The coated glass coverslips were allowed to
dehydrate on a hotplate for a minimum of 2 h set at a temperature
of 40 °C. At the end of the process, the coverslip appeared flat
and clear.
on glass coverslips (Corning, 22 mm × 22 mm) were prepared by
applying 300 μL of 1 wt % (w/w) polymer on a coverslip and evenly
spreading the solution with the side of a pipette tip.66 (link) The coated glass coverslips were allowed to
dehydrate on a hotplate for a minimum of 2 h set at a temperature
of 40 °C. At the end of the process, the coverslip appeared flat
and clear.
8
Fluorescent Microscopy of Actin Cytoskeleton
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BME26 cells (5 × 105 cells/well) were plated on glass coverslips (Corning® Costar®, Cambridge, MA, USA) in 24-well plates, and incubated in complete medium to attach at 34 °C for 24 h. Chemical inhibitors were added at the final concentrations indicated, and 0.05% DMSO was used in negative control wells. After 24 h of treatment, the cells were washed with 0.15 M NaCl, 10 mM sodium phosphate, pH 7.2 (PBS), and immediately fixed on a buffered (PBS) formaldehyde 4% solution for 15 min at room temperature (RT). The cells were then incubated for 20 min (RT) with 200 µL of a solution containing the nuclear marker DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride, molecular Probes, D1306-1 µg/mL), and 1 μL of the F-actin probe phalloidin (Alexa Fluor® 555, Molecular Probes, A34055-300 units). The cells were visualized using a Leica DMI4000 inverted fluorescent microscope equipped with two A4 (DAPI) filter cubes and N2.1 (Phalloidin). Pictures were obtained with a DFC365 FX camera, and images were mounted into a single file with the help of Adobe Photoshop (CC19.0).
9
Immunofluorescence Microscopy of Cultured Cells
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Cells cultured on glass coverslips (Corning) were washed with PBS and fixed using 4% PFA and permeabilized using 0.25% Triton X-100. For imaging after sorting, cells were suspended in PBS at concentrations of ~ 5 × 106 cells/ml and fixed to coverslips by centrifugation. After blocking in 5% BSA, cells were incubated with primary antibodies at 4 °C, washed with PBS, and incubated with highly cross-adsorbed, AlexaFluor-conjugated secondary antibodies at room temperature. Coverslips were mounted to slides using Vectashield hard-set mounting medium with DAPI (Vector Laboratories). Images were acquired using a PerkinElmer UltraVIEW ERS spinning disc confocal imager mounted on a Zeiss AxioVert 200 inverted microscope equipped with a 63×/1.4 Oil DIC Plan-Apochromat 0.19/0.17. Primary antibodies used for fluorescence microscopy were: TFAM (Cell Signaling), cyclin B1 [GNS1] (SCBT), 8-oxo-dG [15A3] (SCBT), COXIV (Cell Signaling), and MAP LC3β [G-9] (SCBT).
10
Live-cell Visualization of L-WNK1 and KS-WNK1
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To visualize L and KS-WNK1 trafficking simultaneously in live cells, eGFP and mRuby were C-terminally tagged to L-WNK1 and KS-WNK1, respectively. HEK-293 cells were transfected as described above in cell culture. Before imaging, cells were transferred to glass coverslips (Corning). Cells were imaged at 60× magnification (Nikon CFI APO TIRF) using a Nikon Ti-E-2000 inverted confocal microscope described above using the Nikon Perfect Focus System with a 37° heated enclosure in Leibovitz’s L-15 media + 1% FBS. eGFP and mRuby were imaged using 488 nm laser excitation with 525/50 nm emission filter, and 561 nm laser excitation with 620/60 nm emission filter, respectively. Images were acquired on an iXon 897 Ultra back-illuminated camera (Andor Technology) with 100 ms exposures every 200 ms for 12 s, and/or IQ2 software was used for image acquisition and postimaging processing.
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