Trypomastigotes (2 × 106/well in 96 well plates) were washed with PBS and incubated with acridine orange dye (Thermo Fisher Scientific) at 4 µM for 5 min in the dark. Non-internalized probe was removed with PBS. The mixture of compounds 1–4 (fraction D-1) was incubated with the parasite at the EC50 value of 4.9 µg/mL for 120 min. Fluorescence levels were measured using Multimode FilterMax F5 (Molecular Devices) microplate reader with excitation and emission wavelengths of 485 and 535 nm, respectively. Nigericin (4 µM) was used as positive control and non-treated parasites as negative control39 (link).
Acridine orange dye
Manufactured by Thermo Fisher Scientific
Sourced in United States
Acridine orange dye is a fluorescent stain used in microscopy and flow cytometry applications. It is a metachromatic dye that can bind to both DNA and RNA, emitting different colored fluorescence based on the type of nucleic acid it is bound to. The core function of acridine orange dye is to enable the visualization and differentiation of nucleic acids in biological samples.
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Lab products found in correlation
4 protocols using acridine orange dye
1
Acridine Orange-Based Parasite Viability Assay
2
Detecting Autophagy in Prostate Cancer
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Acridine orange dye (A3568, ThermoFisher Scientific) was used to evaluate the formation of AVOs in the PCa cells. Briefly, 3 × 104 cells cultured in a μ-Slide 4 Well chamber (ibidi) were treated with DTX for 16 h, stained with acridine orange (1 μg/mL), and covered in a complete medium containing 10% FBS. The formation of AVOs in cells was detected using the EVOS FL Auto Imaging System (Thermo Fisher Scientific). For the detection of GFP-LC3 puncta, PCa cells were transfected with pSELECT-GFP-LC3 plasmids (#10K02-MM, InvivoGen) to stably express the GFP-LC3 fusion gene and maintained under Zeocin (ant-zn-05, InvivoGen) selection in RPMI-1640 with 10% FBS at 37 °C in 5% CO2. DU145 clones with different expression levels of RAGE were treated with DTX for 16 h. GFP-LC3 puncta was imaged using a confocal microscope (Leica Microsystems).
3
Acridine Orange Apoptosis Assay
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Cell apoptosis was analyzed using Acridine Orange dye (AO, Invitrogen, Waltham, MA, USA) [58 (link)]. Thirty nauplii (per replica) were washed in PBS and incubated with 20 µL of 5 µg/mL AO for 20 min at room temperature. After washing in PBS, the nauplii were observed under a fluorescence microscope (Nikon eclipse Ci). The captured images were analyzed using Nis Element 5.20 software through which regions of interest (ROIs) were created to evaluate the fluorescence intensity of equal areas in different larvae. Mean intensity values less than 15 Arbitrary Units (A.U.) were considered negative (mean intensity of control) and higher values were considered positive [59 (link)].
4
Fetal Liver Immunostaining Protocol
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Mouse embryos were harvested at 14.5 days post conception. Fetal livers were dissected and stored in 4% paraformaldehyde at 4°C overnight. Samples were dehydrated in 30% sucrose for 8 hours and embedded in OCT. Cryosections were collected at a thickness of 10 microns. Slides were washed three times in PBS + 0.1% Triton, blocked with 5% normal goat serum and 1% bovine serum albumin for two hours, and stained overnight with rabbit anti-c-Kit (Cell Signaling Technology #3074, 1:400) and rat anti-F4/80 (abcam ab6640, 1:100). Slides were washed five Acridine Orange dye (Invitrogen A3568) was used at a final concentration of 3μg/mL in E3 embryo media. Embryos were incubated with dye for 30 minutes, washed twice with E3, and mounted for imaging.
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