Primary fibroblasts were grown in 35‐mm glass bottom dishes (Sangon Biotech, China). Cells were incubated with MitoTracker® Red (Invitrogen, USA) and DAPI Reagent (Sigma, USA) and then were directly observed under a confocal microscope (Leica, Germany).
Dapi reagent
Manufactured by Merck Group
Sourced in United States
DAPI (4',6-diamidino-2-phenylindole) is a fluorescent dye used to stain nucleic acids, particularly DNA. It is a core component utilized in various microscopy techniques to visualize and identify cell nuclei.
Automatically generated - may contain errors
Lab products found in correlation
Mitotracker red, by Thermo Fisher Scientific (2 mentions)
35 mm glass bottom dishes, by Sangon (2 mentions)
Confocal microscope, by Leica (1 mentions)
Click it aha, by Thermo Fisher Scientific (1 mentions)
Axioplot imager, by Zeiss (1 mentions)
Cck 8 reagent, by Biosharp (1 mentions)
Edu solution, by Solarbio (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Confocal microscope, by Zeiss (1 mentions)
Inverted fluorescence microscope, by Olympus (1 mentions)
Eclipse ti, by Nikon (1 mentions)
10 protocols using dapi reagent
1
Visualizing Mitochondria in Fibroblasts
2
Measuring Protein Translation in Gut
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To assess the levels of protein translation in susceptible and resistant guts, we used the Click-iT AHA (L-azidohomoalanine) for Nascent Protein Synthesis commercial kit (Invitrogen). Flies were orally infected for 16 h as described above, but by adding AHA reagent at 50 μM as final concentration to the infection mix. Guts were then dissected in 1X PBS Triton 0.3%, fixed for a minimum of 30 min in PBS 4% paraformaldehyde, and finally washed with PBS Triton 0.3%. DAPI reagent (Sigma) was used to stain DNA. The R2 region56 (link) of the gut was visualized with an Axioplot imager (Zeiss).
3
Evaluating Cytotoxicity and Proliferation Assays
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For the CCK8 assay, 5000 transfected cells were planted and grown in 96-well plates. When the cells were cultivated for 24, 48 and 72 h, respectively, 10 μ CCK8 reagent (Biosharp, China) was added to the corresponding wells and reacted with the cells for 1 h. Finally, a microplate Reader (SpectraMax ABS, China) was utilized to analyze and record the absorbance at the 450 nm of these cells.
For the colony formation assay, 500 transfected cells were planted and cultured in 6-well plates. After 14 days, the colonies were recorded and counted after the cells were fixed and stained.
For the EdU assay, the transfected cells were immobilized for 15 min with paraformaldehyde after responding for 2 h to the EdU solution (Solarbio, China) of 50 μM at 37 °C, and the DAPI reagent (Sigma, USA) was employed to color the nucleus for 10 min. The representative images of EdU were then captured using a fluorescent microscope (Olympus BX53, Japan), and the EdU positive cells ratio was evaluated as the ratio of red versus blue fluorescent cells.
For the colony formation assay, 500 transfected cells were planted and cultured in 6-well plates. After 14 days, the colonies were recorded and counted after the cells were fixed and stained.
For the EdU assay, the transfected cells were immobilized for 15 min with paraformaldehyde after responding for 2 h to the EdU solution (Solarbio, China) of 50 μM at 37 °C, and the DAPI reagent (Sigma, USA) was employed to color the nucleus for 10 min. The representative images of EdU were then captured using a fluorescent microscope (Olympus BX53, Japan), and the EdU positive cells ratio was evaluated as the ratio of red versus blue fluorescent cells.
4
Nuclei Staining with DAPI
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For nuclei staining, DAPI reagent (Sigma, St. Louis, MO) was used. Briefly, N. benthamiana transformed leaf segments were cut from the plant and then incubated for 5 min in a water solution containing 5 μg/mL of DAPI.
5
EdU Incorporation and DNA Labeling
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EdU solution was diluted and heated in advance as per the guidebook of a cell proliferation imaging analysis kit (Life‐iLab biotech). The treated EdU reagent was then added to each well for 2 h, and EdU residue that was not incorporated with DNA was removed by washing cells using phosphate buffer solution. The cells were subjected to fixation and penetration, followed by incubation with dye reaction solution and DAPI reagent (Sigma). Cell samples were analyzed using fluorescence microscope (Olympus).
6
Visualizing Exosome Internalization in ADSCs
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To observe the internalization of exosomes, we probed the ADSCs-derived exosomes by DiLC18 (Sigma, USA) following manufacturer’s instruction. In short, ADSCs were hatched with DiL-labelled exosomes (DiL-exo) for 24 hours, then the nuclei were dyed by DAPI reagent (Sigma). The ADSCs hatched with PBS were used as control. Images were taken by Carl Zeiss confocal microscope (USA).
7
Immunofluorescence Staining of Oct4
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Cells were fixed in 4% paraformaldehyde and incubated for 1 h in blocking buffer (PBS, 10% FBS, and 0.1% Triton X-100). Primary antibody Oct4 (Santa Cruz Biotechnology, Santa Cruz, CA) was diluted to 1 : 500 in the blocking buffer and was applied overnight at 4°C. After three washes in PBS, secondary antibodies that had conjugated to FITC (fluorescein isothiocyanate) (Vector, Burlingame, CA) were diluted to 1 : 200 in blocking buffer and were applied for 1 h at room temperature. The cells were washed at least three times in PBS and visualized on an Olympus inverted fluorescence microscope. For nuclear counter staining, nuclei were stained with DAPI reagent (Sigma).
8
UVB-Induced Cellular Stress Response
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HaCaT cells were seeded at a density of 4 × 105 cells/mL in a 12-well plate containing previously sterilized, round cover glass slips. After 24 h, cells were treated with loliolide (0–100 µM) for 30 min, washed with PBS, and exposed to UVB (30 mJ). Cells were washed twice with PBS and fixed with 1 mL of 3.7% paraformaldehyde in PBS for 10 min. Cells were washed with PBS two more times, stained with DAPI reagent (Sigma-Aldrich Chemical Co.) (1 µL/mL) for 30 min, and then washed with PBS two more times. The cover slip was then mounted on a rectangular glass slide using mounting solution and left to dry at room temperature for 24 h [35 (link)]. Samples were subsequently examined using a Nikon Eclipse Ti fluorescence microscope (Nikon, Tokyo, Japan). The number of damaged cells in three different pictures was quantified using ImageJ, and afterwards the data were summarized in a plot.
9
Holotomographic Imaging of Mitochondria
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Morphological changes in the cells caused by the test compounds individually and their mixtures were assessed by holotomographic microscopy (HTM). To visualise mitochondrial structures, the cells were incubated with MitoView (Biotium) dye at 100 nM, while nuclei were stained with DAPI reagent (1: 1000, Sigma). The cells were incubated with the above-mentioned dyes for 15 min at 37 °C. After staining the cell structures, observations of the cells were carried out under a holotomographic microscope.
10
Visualizing Mitochondria in Fibroblasts
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Primary fibroblasts growing in 35-mm glass bottom dishes (Sangon Biotech, China). Cells were incubated with MitoTracker® Red (Invitrogen, USA) and DAPI Reagent (Sigma, USA) and then were directly observed under a confocal microscope (Leica, Germany).
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