Exo red exosome rna fluorescent label kit

Parasite EVs were labeled using the Exo-Red exosome RNA fluorescent label kit (System Biosciences). A 180μg protein sample of EVs was incubated for 30 minutes at 37°C with 50μl of Exo-Red dye. Exo-spin columns were used to clean the EV stained preparation and remove excess dye (Cell Guidance Systems, St. Louis MO). 44μl of the stained EV preparation was added to 1x10⁶ THP-1 cells in 1mL of cell culture media (RPMI 1640, 10% FBS, 1% HEPES, 1% L-glutamine, 1% penicillin/streptomycin, 1% Sodium pyruvate, 1% Sodium bicarbonate). The cells and EVs were incubated for 10 minutes, 60 minutes, or 24 hours. At the end of the incubation, the cells were immediately washed with excess volume of Stain Buffer (FBS) (BD Biosciences, San Jose CA). The wash step was repeated three times. The cells were then resuspended in Stain Buffer (FBS) and analyzed on a BD LSRFortessa using BD FACSDiva Software. The acquired data was then analyzed using FlowJo software. The assay was repeated at least once.

For exosome treatment, exosomes were injected into the tail veins of 4-week-old male NOD/SCID/IL2Rγ null mice (4 mice, 5μg exosome per mice), and the mice were sacrificed 24 h later for tissue sections. For in vivo fluorescent imaging, Exo-Red-labeled exosomes (Exo-Red exosome RNA fluorescent label kit, #EXOR100A-1, System Biosciences) isolated from HT-29 or THP-1 cells (50μg of total protein) were injected intravenously into five-week-old male nude mice (BALB/c nu/nu), and the red fluorescence of the whole body was acquired by IVIS spectrum (Caliper Life Sciences). The mice were sacrificed 24 h later, organs prepared and the red fluorescence was quantified in brain, liver, heart, kidney, spleen, lung, stomach, and intestine. Radiant efficiency was measured using Living Image 3.1 software (Caliper Life Sciences).