S2 cells were imaged in concanavalin A–coated (0.25 mg/ml; EMD Millipore) glass-bottom dishes (Ibidi) at 25°C in Schneider’s insect medium (Gibco) + 10% FBS (Invitrogen) using a 100× 1.4 NA Plan-Apochromatic differential interference contrast (DIC) objective lens (Carl Zeiss) mounted on an inverted microscope (TE2000U; Nikon) equipped with a CSU-X1 spinning-disk confocal head (Yokogawa Electric Corporation) and with two laser lines (488 nm and 561 nm). Images were detected with an iXonEM+ EM-CCD camera (Andor Technology), using the NIS-Elements software (Nikon). HeLa cells were imaged in the same system at 37°C in L15 medium + 10% FBS (Invitrogen). Images were analyzed in ImageJ, processed (contrast adjustments) in Photoshop CS4 (Adobe), and represent maximum intensity projections of multiple z stacks (step size: 0.5–1 µm).
Concanavalin a coated
Manufactured by Merck Group
Sourced in Germany, United States
Concanavalin A–coated is a type of lab equipment used in various research applications. It is a lectin, a protein that binds to specific carbohydrate structures. The core function of Concanavalin A–coated is to facilitate the detection, separation, or immobilization of glycoproteins and other carbohydrate-containing molecules in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Schneider s insect medium, by Thermo Fisher Scientific (2 mentions)
Revolution xd, by Oxford Instruments (2 mentions)
Iq live cell imaging software, by Oxford Instruments (2 mentions)
Ixon 897 e, by Oxford Instruments (2 mentions)
Lab tek chamber, by Thermo Fisher Scientific (2 mentions)
Ixonem em ccd camera, by Oxford Instruments (1 mentions)
L 15 medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Nis elements software, by Nikon (1 mentions)
Te2000 u, by Nikon (1 mentions)
Csu x1 spinning disk confocal head, by Yokogawa (1 mentions)
Yol1 34, by Bio-Rad (1 mentions)
Autoquant x, by Media Cybernetics (1 mentions)
Fetal bovine serum (fbs), by Developmental Studies Hybridoma Bank (1 mentions)
Alexa fluor 568 and 647, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Adobe (1 mentions)
Axio imager z1, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Orca r2, by Hamamatsu Photonics (1 mentions)
Glass coverslip, by Corning (1 mentions)
5 protocols using concanavalin a coated
1
Imaging S2 and HeLa Cells with Confocal Microscopy
2
Imaging Cellular Structures in S2 Cells
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S2 cells transiently expressing CD8-GFP were grown on concanavalin A–coated (0.5 mg/ml; EMD Millipore) glass coverslips at 25°C in Schneider’s insect medium (Gibco) + 10% FBS (Invitrogen) and fixed with 4% paraformaldehyde for 10 min and subsequently extracted with 0.1% Triton X-100 for 10 min. After short washes in PBS and blocking with 10% FBS, cells were incubated with mouse anti-lamin Dm0 (clone ADL67, 1:50; Developmental Studies Hybridoma Bank) and rat anti–α-tubulin (YOL1/34, 1:100; Serotec), followed by short washes in PBS and incubation with Alexa Fluor 568 and 647 (1:1,000; Invitrogen). DNA was counterstained with DAPI (1 µg/ml; Sigma-Aldrich) before coverslips were mounted in 90% glycerol + 10% Tris, pH 8.5, + 0.5% N-propylgallate on glass slides. Images were acquired on an AxioImager Z1 (100×, Plan Apochromatic oil DIC objective lens, 1.4 NA; all from Carl Zeiss) equipped with a charge-coupled device (CCD) camera (ORCA-R2; Hamamatsu Photonics) using the Zen software (Carl Zeiss) and blind deconvolved using Autoquant X (Media Cybernetics). Images were processed (contrast adjustments) in Adobe Photoshop CS4 (Adobe) and represent either maximum intensity projections of deconvolved z stacks (step size: 0.22 µm; DAPI; α-tubulin) or a single slice (CD8-GFP; lamin Dm0).
3
Locust Primary Brain Cell Culture
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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.
4
Colocalization Analysis of Cellular Structures
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After incubation in starvation medium for 1.5 h, ∼0.05 OD600nm of cells were plated in starvation medium on Concanavalin A–coated (Sigma-Aldrich) Lab-Tek chambers (Thermo Fisher Scientific) and were allowed to settle for 30 min at 25°C. Then, whole cell z stacks with a step size of 0.3 µm were acquired using a spinning-disk confocal microscope (Revolution XD; Andor Technology) with a Plan Apochromat 100× 1.45 NA objective lens equipped with a dual-mode electron-modifying charge-coupled device camera (iXon 897 E; Andor Technology) and controlled by the iQ Live Cell Imaging software (Andor Technology). Colocalization coefficients were calculated with ImageJ 1.45r and the JACoP plugin (Bolte and Cordelières, 2006 (link)).
5
Visualization of Starvation-Induced Cell Dynamics
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After incubation in starvation medium for 20 min, ~0.05 OD600 nm of cells were plated in starvation medium on Concanavalin A–coated (SIGMA-Aldrich) Lab-Tek chambers (Thermo Fisher Scientific, Waltham, MA, USA) and were allowed to settle for 20 min at 25°C. Due to issues of bleaching, fields of cells were continuously imaged up to 10 min throughout starvation. Whole cell Z stacks with a step size of 0.3 μm were continuously acquired (10 s frames) using a spinning-disk confocal microscope (Revolution XD; Andor Technology, Belfast, United Kingdom) with a Plan Apochromat 100× 1.45 NA objective lens equipped with a dual-mode electron-modifying charge-coupled device camera (iXon 897 E; Andor Technology) and controlled by the iQ Live Cell Imaging software (Andor Technology). Processing was performed with ImageJ 1.47n software.
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