Anti-β-actin (catalog no. 4970) was purchased from Cell Signaling Technology (MA, USA). Mouse monoclonal anti-Rho 1D4 antibody was purified from hybridoma cells74 (link)–76 (link). Alexa Fluor 647-conjugated goat anti-mouse IgG antibody (catalog no. A21236) was from Invitrogen (CA, USA). DAPI fluorescent dye (catalog no. 62248) was purchased from Thermo Fisher (MA, USA). All detergents were purchased from Anatrace (OH, USA). Hydroxylamine hydrochloride (catalog no. 159417) was purchased from Sigma-Aldrich (MI, USA). 9-cis-retinal and 11-cis-retinal were produced by photoisomerization of all-trans-retinal using 420 nm light and purified by normal-phase HPLC using a Phenomenex Luna silica column (catalog no. 00G-4091-P0-AX) and an isocratic gradient of 10% ethyl acetate in hexanes77 (link),78 (link).
Dapi fluorescent dye
Manufactured by Thermo Fisher Scientific
Sourced in United States
DAPI (4',6-diamidino-2-phenylindole) is a fluorescent dye commonly used in molecular biology and cell biology applications. It is a blue-fluorescent dye that binds strongly to the minor groove of DNA. DAPI can be used to stain and visualize cell nuclei and DNA.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Luna silica column, by Phenomenex (1 mentions)
Alexa fluor 647 conjugated goat anti mouse igg antibody, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Hydroxylamine hydrochloride, by Merck Group (1 mentions)
Ki67 antibody, by Thermo Fisher Scientific (1 mentions)
Intrasure kit, by BD (1 mentions)
Pkh26 red fluorescent cell linker, by Merck Group (1 mentions)
Axio observer 5, by Zeiss (1 mentions)
Zen black 2012, by Zeiss (1 mentions)
Immunohistomount, by Santa Cruz Biotechnology (1 mentions)
Vibrant multicolor cell labeling kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using dapi fluorescent dye
1
Purification and Characterization of Rho 1D4 Antibody
2
Cell Cycle Analysis by Flow Cytometry
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For detection of cell cycle distribution upon different treatments, cells were trypsinized, washed with PBS, and centrifuged for 5 min at 250× g. BD IntraSure kit (BD Biosciences) was used for cell fixation and permeabilization, according to manufacturer’s protocol and followed by 15 min of dark incubation in room temperature of cells with Ki-67 antibody (Thermo Fisher Scientific Inc., Waltham, MA, USA). Thereafter, cells were stained with DAPI fluorescent dye (excitation maximum at 360 nm and fluorescence emission maximum at 460 nm, Thermo Fisher Scientific Inc.) and processed for FACS-based cell cycle analysis (Figure S2 ).
3
Fluorescent Labeling of Marine EVs
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To track the marine EVs, the surface membrane was labeled with PKH-26 red fluorescent cell linker (Sigma-Aldrich) for 24 h at 4 °C. Subsequently, the remaining PKH-26 dye was washed three times using PBS and 100 kDa filter and diluted in serum-free RPMI-1640 containing AA to treat RAW-264.7 cells for 12 h at 37 °C under 5% CO2. After 12 h, the sample medium was changed to DAPI fluorescent dye (Invitrogen, Waltham, MA, USA) for 15 min and then washed twice with PBS. Endocytosis was observed using a fluorescence microscope (Axio-Observer 5; Carl Zeiss, Oberkochen, Germany).
4
Vesicle Uptake in Cancer Cells
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MCF-7 breast adenocarcinoma cells and HCT-15 colon adenocarcinoma cells were cultured on a 96-well plate on glass coverslips in the amount of 20,000 cells per well and cultured for 24 h. Vesicles were obtained from MSCs and SNB-19 cells were preliminary stained with DiD vital dye (Vibrant Multicolor Cell-Labeling Kit, Invitrogen, Waltham, MA, USA) according to the previously described protocol.
Vesicles were added to the cells at a concentration of 2 µg per 100 µL (20 µg/mL) and cultured for 4 h. After washing three times for 5 min in PBS, cells were stained with DAPI fluorescent dye (4′,6-diamidino-2-phenylindole; dilution 1:50,000 in TBS; Invitrogen, Waltham, MA, USA) for 7 min, and washed again. Coverslips were mounted on the slides with a mounting medium (ImmunoHistoMount, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The samples were investigated under a LSM 780 confocal microscope (Carl Zeiss, Jena, Germany) using Zen black 2012 software (Carl Zeiss, Jena, Germany). All samples were imaged in the z-plane using identical confocal settings (laser intensity, gain, and offset).
Vesicles were added to the cells at a concentration of 2 µg per 100 µL (20 µg/mL) and cultured for 4 h. After washing three times for 5 min in PBS, cells were stained with DAPI fluorescent dye (4′,6-diamidino-2-phenylindole; dilution 1:50,000 in TBS; Invitrogen, Waltham, MA, USA) for 7 min, and washed again. Coverslips were mounted on the slides with a mounting medium (ImmunoHistoMount, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The samples were investigated under a LSM 780 confocal microscope (Carl Zeiss, Jena, Germany) using Zen black 2012 software (Carl Zeiss, Jena, Germany). All samples were imaged in the z-plane using identical confocal settings (laser intensity, gain, and offset).
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