TC proliferation was evaluated using the Click-iT EdU Cell Proliferation Kit (ThermoFishe; Waltham, MA, USA). EdU solution was added into media of TCs incubated with conditioned media from either tumor-free or tumor-bearing tissues, after 8 h of incubation cells were fixed and permeabilized for later EdU detection following manufacturer indications. Images were taken by a fluorescence microscope (Zeiss; Jena, Germany). TC cell death by apoptosis was evaluated using the APO-BrdU TUNEL Assay Kit (Invitrogen; Waltham, MA, USA). BrdU solution was added into media of TCs with conditioned media from either tumor-free or tumor-bearing tissues, 24 h later cells were fixed and processed according to manufacture guidelines. Images were taken by a fluorescence microscope (Zeiss; Jena, Germany). Analysis and cell quantification were performed using the cell image analysis software CellProfiler version 4.2.6 [19 (link),20 (link)]. n = 4 with triplicates per condition.
Fluorescence microscope
Manufactured by Zeiss
Sourced in Germany, United States, United Kingdom, China, Italy, Japan, Canada, France, Switzerland
The Zeiss Fluorescence Microscope is an optical instrument used to study the properties of fluorescent materials. It employs specific wavelengths of light to excite fluorescent molecules, causing them to emit light at a different wavelength, which is then observed and analyzed.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (56 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (42 mentions)
Isis 5, by MetaSystems (33 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (28 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (26 mentions)
Bovine serum albumin (bsa), by Merck Group (23 mentions)
Paraformaldehyde (pfa), by Merck Group (20 mentions)
Triton x 100, by Merck Group (19 mentions)
Axiovision software, by Zeiss (13 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (12 mentions)
Hoechst 33342, by Thermo Fisher Scientific (12 mentions)
Dcfh da, by Beyotime (11 mentions)
In situ cell death detection kit, by Roche (11 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (9 mentions)
Hoechst 33342, by Merck Group (8 mentions)
Hoechst 33258, by Merck Group (8 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (8 mentions)
Hoechst 33258, by Beyotime (7 mentions)
Edu assay kit, by RiboBio (7 mentions)
Propidium iodide, by Merck Group (7 mentions)
1 695 protocols using fluorescence microscope
1
Quantifying Cell Proliferation and Apoptosis
2
Measuring Intracellular Ca2+ and ROS Levels
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The intracellular Ca2+ level was measured using the Fluo-4-AM (Invitrogen, United States) dye. PC12 cells were stained with 2.5 μM of Fluo-4-AM solution for 30 min in darkness at 37°C and then washed for three times in Hank’s Balanced Salt Solution (HBSS) (Corning, United States). Green fluorescence, which reflects the intracellular Ca2+ level, was recorded with a fluorescence microscope (Carl Zeiss, Germany).
Intracellular ROS levels were detected using fluorescence microscopy with DCFH-DA (Beyotime Biotechnology, Beijing, China). The DCFH-DA was intracellularly deacetylated by a nonspecific esterase, further oxidized by ROS to produce the fluorescent compound 2,7-dichlorofluorescein (DCF) (An et al., 2017 (link)). The analysis was performed with the assay kit protocol. Briefly, PC12 cells were washed twice in DMEM without FBS and incubated with DCFH-DA at 37°C for 25 min. The cells were then washed twice with PBS and analyzed in a fluorescence microscope (Carl Zeiss, Germany).
The intensity of Ca2+ and ROS-labelled cells was further quantified. The labeled cells were counted from five areas under × 20 field or × 40 field randomly chosen from each well using ImageJ software.
Intracellular ROS levels were detected using fluorescence microscopy with DCFH-DA (Beyotime Biotechnology, Beijing, China). The DCFH-DA was intracellularly deacetylated by a nonspecific esterase, further oxidized by ROS to produce the fluorescent compound 2,7-dichlorofluorescein (DCF) (An et al., 2017 (link)). The analysis was performed with the assay kit protocol. Briefly, PC12 cells were washed twice in DMEM without FBS and incubated with DCFH-DA at 37°C for 25 min. The cells were then washed twice with PBS and analyzed in a fluorescence microscope (Carl Zeiss, Germany).
The intensity of Ca2+ and ROS-labelled cells was further quantified. The labeled cells were counted from five areas under × 20 field or × 40 field randomly chosen from each well using ImageJ software.
3
NF-κB Activation and Cell Viability Assays
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After fixation, cells were incubated with primary antibodies against NF-κB p65 and then secondary antibodies. Cells were costained with DAPI (Beyotime, Shanghai, China) and then visualized using a fluorescence microscope (Zeiss, Jena, Germany) and a laser scanning confocal microscope (Zeiss, Jena, Germany). Cell morphology was detected by Actin-Tracker Green staining kit (Beyotime, Shanghai, China) under an AxioVert A1 fluorescence microscope. Cell activity was detected according to instructions of Calcein AM Cell Viability Assay Kit (Beyotime, Shanghai, China). Dead cells were dyed red and observed under fluorescence microscope (Zeiss, Jena, Germany). The Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed following the manufacturer’s instructions (Beyotime, Shanghai, China).
4
Quantifying Intracellular ROS Generation
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To measure the amount of intracellular ROS generated in cells, cells were treated with or without platycodin D or NAC for 1 h, before another 1 h culture with the addition of H 2 O 2 . The cells were washed with phosphate-buffered saline (PBS), and lysed with PBS containing 1% Triton X-100 for 10 min at 37°C. The cells were then stained with 10 µM 2' ,7'-dichlorofluorescein diacetate (DCF-DA, Eugene, OR, USA) for 30 min at room temperature (RT) in the dark, and washed with PBS. Intracellular ROS generation was immediately recorded at 515 nm by a flow cytometer (Becton Dickinson, San Jose, CA, USA). The results were expressed as the percentage increase relative to untreated cells (Yoon et al. 2019) (link). It was also analyzed the levels in ROS by fluorescence microscopy. To this end, cells cultured in glass cover slips were treated with H 2 O 2 in the absence or presence of platycodin D or NAC. After 1 h of treatment, the cells were incubated in a medium containing 10 µM DCF-DA at 37°C for 20 min. Stained cells were washed twice with PBS and observed with a fluorescence microscope (Carl Zeiss, Oberkochen, Germany) . In same condition, to evaluate the levels of mitochondrial-derived ROS were detected in cells stained for 20 min with 5 µM MitoSOX™ Red (Thermo Fisher Scientific, Waltham, MA, USA). Stained images were acquired with a fluorescence microscope (Carl Zeiss).
5
NF-κB p65 Nuclear Translocation Assay
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The NF-κB p65 nuclear translocalization was detected by an immunofluorescence assay using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany). After designated treatments, the cells were fixed with 3.7% paraformaldehyde (Sigma-Aldrich Chemical Co.) in PBS for 10 min at 4°C, permeabilized with 0.4% Triton X-100 in PBS for 10 min, and blocked with 5% bovine serum albumin for 1 h. The cells were probed with anti-p65 NF-κB antibody (Santa Cruz Biotechnology, Inc.) overnight at 4°C and then incubated with fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) for 2 h at room temperature. The position of the cell nucleus was determined with 4,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich Chemical Co.) solution (1 mg/mL) for 15 min. After washing with PBS, the fluorescence was visualized using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany).
6
Apoptosis and DNA Damage Analysis in NSCLC
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NSCLC cells were treated with 10 mM metformin and/or 25 μM celecoxib for 48 h. Cells were washed twice with PBS and then fixed in 4% (v/v) paraformaldehyde and permeabilized with 0.2% (v/v) Triton X-100 in PBS for 5 min. The TUNEL assay was performed by using a One Step TUNEL Apoptosis Assay Kit (Beyotime Inst Biotech, China). In brief, TUNEL detection solution was added to each sample and incubated at 37°C for 60 min. Then, the genomic DNA of apoptotic cells was broken, and the exposed 3′-OH was catalyzed by terminal deoxynucleotidyl transferase (TdT) to add dUTP labeled by FITC. After washing with PBS, the cells were restained with propidium iodide (PI). The fluorescent photos of the cells were captured by a fluorescence microscope (Zeiss, Jena, Germany). For immunofluorescence staining, antibodies against γ-H2AX (1:500 dilutions; Cell Signaling Technology, MA, USA) were used and then conjugated with Alexa Fluro® 488 goat anti-rabbit IgG (1:2000 dilutions; Invitrogen, Carlsbad, CA). The fluorescent photos of the cells were captured by a fluorescence microscope (Zeiss, Jena, Germany).
7
Apoptosis Assays for Cancer Cell Lines
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Cell apoptosis was determined using the terminal deoxynucleotidyl transferase (TdT) deoxyuridine dUTP nick-end labeling (TUNEL) and Annexin V assays as previously described (Kee et al., 2017 (link)). CT26 and HT29 cells were seeded in six-well plates, treated with G.A for 24 h, fixed with 3.7% paraformaldehyde for 20 min, and then washed with phosphate-buffered saline (PBS). The cells were incubated with 4′,6-diamidino-2-phenylindole (DAPI) solution (2.5 μg/mL) for 3 min at room temperature and washed with PBS. Apoptotic cells and stained nuclei were observed using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany). In addition, the apoptotic cells were analyzed using the MUSE Annexin V and dead cell kit (Millipore, Billerica, MA, United States) in accordance with the recommended protocol. The stained cells were analyzed using the Muse cell analyzer.
8
Quantifying Apoptosis via TUNEL Assay
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Cell apoptosis was tested by the TUNEL staining assay. At least 300 cells per treatment in five-random views under fluorescence microscope (Zeiss, Shanghai, China, 1 to 100) were included to calculate the TUNEL-nuclei percentage [42 (link)]. Nuclei were visualized using Hoechst 33342 dye (Sigma).
9
Fluorescent Mice and Cell Line Protocols
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HepG2 was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, 4T1 was purchased from the University of Pittsburgh Medical Center, and DEX was purchased from Anhui Fengyuan Pharmaceutical Co., Ltd. Fluorescent nude mice and BALB/c mice were provided by the Experimental Animal Center of Soochow University. Individual ventilated caging (IVC) mice were independently supplied with air. The feeding system was purchased from Soochow Suhang Equipment Co., Ltd.; DMEM medium and RPMI-1640 medium were purchased from HyClone, Logan, UT, USA; the fluorescence microscope was purchased from Zeiss, Oberkochen, Germany; fluorescent lights and lenses were purchased from Nightsea, Lexington, MA, USA; the frozen slicer was purchased from Leica, Buffalo Grove, IL, USA; and the flow cytometer was purchased from Beckman CytoFLEX, Krefeld, Germany.
10
Peroxynitrite Production Imaging
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