A549 cells cultivated in a Nunc™ Petri dish (Thermo Fisher Scientific, Inc.) were washed three times with 1X PBS, fixed in 4% paraformaldehyde for 20 min at 4˚C and washed again three times with PBS. The cells were then permeabilized with 0.2% Triton X-100 in PBS and blocked for 30 min at 4˚C with 3% bovine serum albumin (Thermo Fisher Scientific, Inc.). The cells were then incubated overnight at 4˚C with anti-LC3 primary antibodies (1:400 dilution; SAB4300571, Sigma-Aldrich; Merck KGaA) in blocking buffer. After washing with PBS, the cells were incubated with Alexa Fluor 555 fluorescent secondary antibody (1:100; cat. no. bs-0295G-AF555; BIOSS) for 60 min at room temperature. The nuclei were then stained with 4',6-diamidino-2-phenylindole at 1:5,000 dilution for 5 min at room temperature. The cells were washed twice with PBS and fluorescence images were captured using a confocal microscope (Smartproof 5; Carl Zeiss AG). The intensity of staining was determined by measuring the integrated optical density (IOD) in 10 different fields for each sample.
Nunc petri dish
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Nunc™ Petri dish is a versatile and reliable laboratory equipment used for culturing and studying microorganisms, cell lines, and other biological samples. It provides a controlled environment for these specimens to grow and thrive. The Nunc™ Petri dish is made of high-quality, transparent material that allows for easy observation and analysis of the samples.
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Lab products found in correlation
3 protocols using nunc petri dish
1
Immunofluorescence Staining of LC3 in A549 Cells
2
Immunofluorescence Staining of A549 Cells
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The A549 cells were grown on Nunc™ Petri dish (Thermo Fisher, Waltham, MA, USA) and then fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) buffer for 30 min. After washing with PBS, the cells were permeabilized with 0.1% Triton X-100 in PBS buffer for 15 min and then blocked with 1% bovine serum albumin (BSA) (Thermo Fisher, Waltham, MA, USA) for 30 min. After blocking, the cells were incubated (4°C overnight) with rabbit anti-FLAG antibody (Cell Signaling Technology, Boston, USA), which was used as a primary antibody. The cells were then washed with PBS and incubated with fluorescein isothiocyanate- (FITC-) conjugated fluorescent antibody (Bioss, Beijing, China) for 1 h at room temperature. The nuclei of the cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (Bioss, Beijing, China) for 5 min and visualized using a confocal laser scanning microscope (Leica, Germany).
3
Isolation and Culture of Human Osteoblasts
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Human cancellous bone samples were dissected into approximately 1–3 mm3 bone tissue blocks within 2–5 h of collection. The cartilage/soft tissue was completely removed. This was followed by repeated PBS (4 mL) washing until neither fat droplets nor erythrocytes were visible in the supernatant. 10 cancellous bone cubes were placed in a Petri dish (Thermo Scientific™ Nunc™ Petri dish, Thermo Fisher Scientific, Waltham, MA, USA) and mixed with complete cell culture medium. The medium was changed at two-day intervals. After reaching cell confluence, enzymatic detachment of hOB from the bottom of the Petri dish was achieved using 0.25% trypsin in calcium-free PBS, in order to transfer them to cell culture flasks (SARSTEDT AG und Co. KG, Nümbrecht, Germany). After 14 days of cultivation, 106 hOB/mL were used for the experiments.
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