Amniotic cells, glass-bottom 35 mm u-dishes (Ibidi GmbH, Germany) coated with fibronectin, were co-transfected with H2B-GFP and pmRFP-tubulin expression plasmids (Addgene, MA, USA) using Lipofectamine 3000 transfection reagent and according to the manufacturer's instructions. Live cell imaging was performed 48 hr following transfection under a spinning-disk confocal system Andor Revolution XD (Andor Technology, UK) coupled to an Olympus IX81 inverted microscope (Olympus, UK) equipped with an electron-multiplying CCD iXonEM Camera and a Yokogawa CSU-22 unit based on an Olympus IX81 inverted microscope. Two laser lines at 488 and 561 nm were used for the excitation of GFP and pmRFP and the system was driven by IQ software (Andor Technology, UK). Z-stacks (0.8–1.0 μm) covering the entire volume of the mitotic cells were collected every 1.5 min with a PLANAPO 60×/1.4 NA objective. ImageJ was used to process all the videos.
Andor revolution xd
Manufactured by Oxford Instruments
Sourced in United Kingdom
The Andor Revolution XD is a high-performance scientific imaging system designed for advanced microscopy applications. It features a back-illuminated EMCCD camera sensor that provides high quantum efficiency, low noise, and fast readout rates. The system is capable of capturing high-resolution images and videos with minimal exposure times, making it suitable for a range of scientific disciplines.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Merck Group (4 mentions)
Streptomycin, by Merck Group (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Prmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Csu 22 unit, by Olympus (2 mentions)
Ix81 inverted microscope, by Olympus (2 mentions)
Iq software, by Oxford Instruments (1 mentions)
Glass bottom 35 mm u dishes, by Ibidi (1 mentions)
Lipofectamine 3000, by Addgene (1 mentions)
Ixon ultra 897 ccd camera, by Oxford Instruments (1 mentions)
Csu x1 confocal scanner, by Yokogawa (1 mentions)
Andor iq3, by Oxford Instruments (1 mentions)
Axio observer microscope, by Zeiss (1 mentions)
Zen software, by Zeiss (1 mentions)
No 1.5h, by Marienfeld (1 mentions)
Orca flash 4.0 camera, by Hamamatsu Photonics (1 mentions)
Ixonem camera, by Yokogawa (1 mentions)
7 protocols using andor revolution xd
1
Live-cell Imaging of Mitotic Cells
2
Chicken Macrophage Immune Response to MOMVs
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The HD11 cells, a transformed chicken macrophage cell line, were used to investigate whether MOMVs could induce innate immune responses in vitro. We first explored the uptake of MOMVs by HD11 macrophages using a co-culture experiment as described previously [17 (link)]. Briefly, dialkylcarbocyanine iodide (DiI, Sigma-Aldrich)-labeled MOMVs were co-cultured with HDl1 cells in complete PRMI-1640 medium (Gibco) containing 10% heat-inactivated FBS (HyClone) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin, Sigma-Aldrich) at 37 °C in a 5% CO2 atmosphere. After incubation, the cell nucleus was stained with 4, 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) and then visualized with High-speed spinning-disk confocal microscope (Andor Revolution XD, Andor Technology, UK). The cells that were not treated with MOMVs were used as the control. We next performed a stimulation assay to evaluate the immune responses of chicken macrophage to MOMVs. HD11 monolayers (1 × 106 cells/mL) were cultured with various doses of MOMVs (0–100 ng/mL) in cell culture medium described above. After 16-h stimulation, the cell culture supernatants were collected for determining the production of cytokines.
3
Imaging Centrosomes and Nuclei in C. elegans Embryos
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Adult gravid hermaphrodite worms were dissected in a watch glass filled with Egg Salts medium (118 mM KCl, 3.4 mM MgCl2, 3.4 mM CaCl2 and 5 mM HEPES pH 7.4), and embryos were mounted on a fresh 2% agarose pad and covered with an 18 mm×18 mm coverslip (No. 1.5H, Marienfeld). Embryos co-expressing GFP::histone H2B and GFP::γ-tubulin for tracking of centrosomes and nuclei (Fig. 1 F–H; Fig. 2 C–G; Fig. 3 G) were imaged on an Axio Observer microscope (Zeiss) equipped with an Orca Flash 4.0 camera (Hamamatsu), a Colibri.2 light source, and controlled by ZEN software (Zeiss). All other imaging was performed on a Nikon Eclipse Ti microscope coupled to an Andor Revolution XD spinning disk confocal system composed of an iXon Ultra 897 CCD camera (Andor Technology), a solid-state laser combiner (ALC-UVP 350i, Andor Technology), and a CSU-X1 confocal scanner (Yokogawa Electric Corporation), controlled by Andor IQ3 software (Andor Technology). All imaging was performed in temperature-controlled rooms kept at 20°C. Time-lapse sequences were processed and analyzed with Fiji software (Image J version 2.0.0-rc-56/1.51 h).
4
Internalization and Immunomodulation of Lipid-rich EVs in Chicken Macrophages
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HD11 cells, a transformed chicken Mφ cell line derived from bone marrow [30 (link)], were used in this study and cultured in the complete PRMI-1640 medium (Gibco, #22400089) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Zeta-Life, #Z7181FBS-500), 100 U/mL penicillin and 100 μg/mL streptomycin (Sigma, #P4333) in an atmosphere of 5% CO2 at 37 °C. Visualization of the internalization of LrEVs by HD11 cells was performed as presented in our previous study [27 ]. Briefly, HD11 cells (5 × 105 cells/mL) were co-incubated with DiI (Sigma, #42364)-labeled LrEVs (10 μg/mL) for 6 h. After washing and staining with 4′, move to the same line (DAPI; Sigma, #D9542), the cells were detected using the high-speed spinning-disk confocal microscope (Andor Revolution XD, Andor Technology, UK).
For the LPS-challenged Mφ assay, HD11 cells (5 × 105 cells/mL), were pretreated with PBS or LrEVs (10 μg/mL) for 12 h and stimulated with PBS or LPS (1 μg/mL) for 12 h. The cells were collected for the determination of cytokine gene expression, NF-κB p65 transcription factor activity and cell viability. Three independent experiments were performed per treatment.
For the LPS-challenged Mφ assay, HD11 cells (5 × 105 cells/mL), were pretreated with PBS or LrEVs (10 μg/mL) for 12 h and stimulated with PBS or LPS (1 μg/mL) for 12 h. The cells were collected for the determination of cytokine gene expression, NF-κB p65 transcription factor activity and cell viability. Three independent experiments were performed per treatment.
5
Spinning-Disk Confocal Imaging of Mitotic Fibroblasts
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Fibroblasts were grown in ibiTreat polymer‐coated 35‐mm μ‐dishes (Ibidi GmbH, Germany) and imaged using the Andor Revolution XD spinning‐disk confocal system (Andor Technology, Belfast, UK), equipped with an electron‐multiplying charge‐coupled device iXonEM Camera and a Yokogawa CSU 22 unit based on an Olympus IX81 inverted microscope (Olympus, Southend‐on‐Sea, UK). The system was driven by Andor IQ software, and laser lines at 488 and 561 nm were used for excitation of GFP and mCherry, respectively. Z‐stacks (0.8–1.0 μm) covering the entire volume of individual mitotic cells were collected every 1.5 min using a PlanApo 60×/1.4 NA objective. ImageJ/Fiji software was used to edit the movies in which every image represents a maximum intensity projection of all z‐planes.
6
MOMV Uptake and Immune Response in Chicken Macrophages
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The HD11 cells, a transformed chicken macrophage cell line, were used to investigate whether MOMVs could induce innate immune responses in vitro. We rst explored the uptake of MOMVs by HD11 macrophages using a co-culture experiment as described previously [17] . Brie y, dialkylcarbocyanine iodide (DiI, Sigma-Aldrich)-labeled MOMVs were co-cultured with HDl1 cells in complete PRMI-1640 medium (Gibco) containing 10% heat-inactivated FBS (HyClone) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin, Sigma-Aldrich) at 37°C in a 5% CO 2 atmosphere. After incubation, the cell nucleus was stained with 4, 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) and then visualized with High-speed spinning-disk confocal microscope (Andor Revolution XD, Andor Technology, UK). The cells that were not treated with MOMVs were used as the control. We next performed a stimulation assay to evaluate the immune responses of chicken macrophage to MOMVs. HD11 monolayers (1 × 10 6 cells/mL) were cultured with various doses of MOMVs (0-100 ng/mL) in cell culture medium described above. After 16-h stimulation, the cell culture supernatants were collected for determining the production of cytokines.
7
Chicken Macrophage Immune Response to MOMVs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The HD11 cells, a transformed chicken macrophage cell line, were used to investigate whether MOMVs could induce innate immune responses in vitro. We first explored the uptake of MOMVs by HD11 macrophages using a co-culture experiment as described previously [17] . Briefly, dialkylcarbocyanine iodide (DiI, Sigma-Aldrich)-labeled MOMVs were co-cultured with HDl1 cells in complete PRMI-1640 medium (Gibco) containing 10% heat-inactivated FBS (HyClone) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin, Sigma-Aldrich) at 37°C in a 5% CO 2 atmosphere. After incubation, the cell nucleus was stained with 4, 6diamidino-2-phenylindole (DAPI, Sigma-Aldrich) and then visualized with High-speed spinning-disk confocal microscope (Andor Revolution XD, Andor Technology, UK). The cells that were not treated with MOMVs were used as the control. We next performed a stimulation assay to evaluate the immune responses of chicken macrophage to MOMVs. HD11 monolayers (1 × 10 6 cells/mL) were cultured with various doses of MOMVs (0-100 ng/mL) in cell culture medium described above. After 16-h stimulation, the cell culture supernatants were collected for determining the production of cytokines.
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