Analysis of germline cell death events were done using the CED-1::GFP reporter. Worm slides were prepared 24 h after recovery from either UVC or drug treatments (and untreated controls). Agar pads were prepared by adding 55 µL to 60 µL of molten 2% agarose, flattening by another slide, and adding 5 µL 3 mM levamisole dissolved in M9 buffer to anesthetize the worms. Worms were picked from seeded NGM plates and placed in the agar pad with levamisole. Normarski (DIC) and green fluorescent images were taken using the Nikon Eclipse Ti-S Fluorescent Microscope at 20× objectives. Images were processed, and cell death events were scored using ImageJ with Bio-Formats plug-in.
Eclipse ti s fluorescent microscope
Manufactured by Nikon
Sourced in Japan
The Eclipse Ti-S fluorescent microscope is a high-performance laboratory instrument designed for imaging and analysis. It features a motorized stage, advanced optics, and a range of fluorescence capabilities to enable detailed observation and study of samples.
Automatically generated - may contain errors
Lab products found in correlation
Cytation 5 multi mode reader, by Agilent Technologies (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Prolong gold antifade with dapi, by Thermo Fisher Scientific (1 mentions)
Pg 21, by Merck Group (1 mentions)
Ab9474, by Abcam (1 mentions)
Ab28364, by Abcam (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Mouse anti rabbit fitc, by Thermo Fisher Scientific (1 mentions)
Scriptcap m7g capping system, by CellScript (1 mentions)
Megascript t7 transcription kit, by Thermo Fisher Scientific (1 mentions)
Lipofectamine messengermax, by Thermo Fisher Scientific (1 mentions)
I scei endonuclease, by New England Biolabs (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Vectashield antifade mounting medium h 1000, by Vector Laboratories (1 mentions)
Ds ri1 camera, by Nikon (1 mentions)
9 protocols using eclipse ti s fluorescent microscope
1
Quantifying Germline Cell Death in C. elegans
2
Comprehensive Analytical Methods for Chemical and Biological Characterization
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NMR spectra were performed by JEOL-ECX500 instrument (Akishima, Japan) or Bruker Biospin AG-400 instrument (Bruker Optics, Germany) using DMSO-d6 as solvent and tetramethylsilane as the internal standard. Melting points were determined on a SGW X-4B microscopic melting point apparatus (Shengguang Instrument Co., Ltd., China) and were uncorrected. HRMS spectra were obtained on Thermo Scientific UltiMate 3000 spectrometer (Waltham, USA) or Waters Xevo G2‐S QTOF MS (Waters MS Technologies, Manchester, UK). Immunofluorescence staining assay was performed by tubulin-tracker red kit (Beyotime Institute of Biotechnology, Shanghai, China) and observed using a Nikon ECLIPSE Ti－S fluorescent microscope (Nikon, Japan). The tubulin polymerisation assay was carried out using the tubulin polymerisation assay kit (cytoskeleton, #BK004P) and recorded by Cytation™ 5 multi-mode readers (BioTek Instruments, Inc. USA). The rhodanine-3-acetic acid and various amine analogues were purchased from Aladdin Industrial Inc. (Shanghai, China) or Bide Pharmatech Co., Ltd. (Shanghai, China). The FACSCalibur™ flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA) was employed to analyse the cell cycle arrest.
3
Immunofluorescence Analysis of Androgen Receptor
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Cells were seeded on coverslips, fixed, permeabilized, blocked, and visualized (anti-AR; RRID:AB_310214; #PG-21, Millipore) and Alexa Fluor 568 (RRID:AB_10563566; #A11036; Molecular Probes) antibodies. Nuclei were visualized using ProLong Gold Antifade with DAPI (#P36941; Thermo Scientific). Cells were viewed under a Nikon Eclipse Ti-S fluorescent microscope and images captured using NIS-Elements software.
4
Immunohistochemical Analysis of AR Expression
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IHC was performed for AR as described (14 (link)). Briefly, cells were seeded on coverslips, cultured, and treated as indicated. Following treatment cells were fixed, permeabilized, and blocked overnight. AR was visualized using primary anti-AR antibody (RRID:AB_307266; #ab9474; Abcam) and CD31 (RRID:AB_726362; #ab28364; Abcam). ProLong Gold Antifade Reagent with DAPI (#P36941, Thermo Fisher Scientific) was used to visualize the nuclei. Cells were viewed under a Nikon Eclipse Ti-S fluorescent microscope and images captured using NIS-Elements software.
5
Immunofluorescence Characterization of Human Fetal Neural Stem Cells
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Human FNSCs were plated onto coverslips coated with poly-D-Lysine. They were washed with PBS and fixed with 2% PFA. Cells were incubated for 1 hour in blocking solution (1% BSA with 0.1% Triton X-100) and then washed with PBS. The cells were incubated overnight at 4˚C with primary antibody (Mouse anti-Nestin 1:1000, Cat no. 33475, CST, MA, USA; Rabbit anti-SOX2 1:1000, Cat no. 23064, CST, MA, USA). The cells were washed thrice with PBS and then incubated with secondary antibody (Mouse anti-Rabbit FITC, 1:1000: Cat no. A11008, Invitrogen; Goat anti-Mouse Alexa Fluor 594, Cat no. A-11005, Invitrogen) for 1 hour at room temperature. Cells were then washed thrice with PBS, and mounted onto glass slides using Vectashield mountant containing DAPI. The slide was allowed to dry overnight.
Images were taken on Nikon Eclipse Ti-S fluorescent microscope (Tokyo, Japan) and analyzed with NIS-Elements BR software.
Images were taken on Nikon Eclipse Ti-S fluorescent microscope (Tokyo, Japan) and analyzed with NIS-Elements BR software.
6
Production of Modified mRNA via IVT
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Template DNA was generated by linearizing the aforementioned plasmids using I-SceI endonuclease (New England Biolabs, Ipswich, MA, USA) before IVT with the MEGAscript T7 Transcription Kit (Life Technologies, Waltham, MA, USA). Post-transcriptional modifications were applied using the ScriptCap m7G Capping System, 2′-O-Methyltransferase Kit, and the A-Plus Poly(A) Polymerase Tailing Kit (CELLSCRIPT, Madison, WI, USA). All mRNAs were purified with the RNeasy Mini Kit (QIAGEN) after IVT and after each modification. For production of modified mRNA, the uridine nucleotide was completely replaced by N1-methylpseudouridine (tebu-bio, Boechout, Belgium) during IVT. Correct translation of EGFP-encoding IVT mRNAs and pDNA was verified after transfection of baby hamster kidney (BHK) cells using Lipofectamine MessengerMAX (Thermo Fisher Scientific) and subsequent evaluation using the Nikon Eclipse Ti-S fluorescent microscope (Nikon, Leuven, Belgium).
7
Immunofluorescent Characterization of Stem Cell Markers
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Expression of β-catenin, CK12, CK19, ABCG2 was qualitatively evaluated with immunofluorescent staining. Cells were fixed in cold absolute methanol for 20 min and washed three times with PBS. Cell membranes were permeabilized for 30 min in 0.2% Triton X-100. Nonspecific binding sites were blocked with 1% BSA in PBS for 1 h. Cells were then stained with primary antibodies diluted with 1% BSA/PBS for 1 h at room temperature (RT) or overnight at 4 °C. Afterwards, cells were stained with secondary antibodies diluted in PBS for 1 h at RT. Nuclei were stained with 1 µg/mL 4, 6-diamino-2-phenylindole (DAPI, Millipore) for 5 min. Then, the cells were mounted with Vectashield® antifade mounting medium (H-1000, Vector Laboratories, Burlingame, CA, USA). Cell images were captured with a Nikon Eclipse Ti-S fluorescent microscope equipped with a DS-Ri1 camera (Nikon Instruments Inc., Tokyo, Japan). The intensity of β-catenin in the nucleus and cytoplasm was measured using CellProfiler software (www.cellprofiler.org , accessed on 12 January 2022).
8
Tubulin Tracking in A549 Cells
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According to the previous protocol48 (link),49 , A549 cells were seeded in 6-well plate and incubated overnight. The cells were incubated with DMSO or various concentrations of target compounds. Then, 6-well plates were kept at 37 °C and 5% CO2 for 48 h. Afterwards, the cells were washed with phosphate-buffered saline (PBS, 10 mM, pH 7.3) and fixed with 4% formaldehyde solution for 15 min, and subsequently washed with 0.1% Triton X-100 in PBS. Subsequently, cells were incubated with diluting tubulin-tracker red for 40 min in a dark environment and washed three times using 0.1% Triton X-100 of PBS, and then stained with DAPI (2 µg/mL). Finally, the results were imaging using a Nikon ECLIPSE Ti-S fluorescent microscope (Nikon Co, Japan).
9
Quantifying THP-1 Cell Adhesion to RASFs
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RASFs were incubated with various concentrations of Antcin K for 24 h at 37°C and then with BCECF-AM-labeled THP-1 cells for 1 h at 37°C. Non-adherent cells were removed by PBS washing. Attached THP-1 cells in RASFs were counted using a Nikon Eclipse Ti-S Fluorescent microscope (Nikon, Japan) with images captured at 10× magnification. Captured images were counted and quantified by MacBiophotonics ImageJ software (v1.51, National Institutes of Health, Bethesda, MD, USA) (19 (link), 20 (link), 27 (link)).
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