Visualizing uptake of small extracellular vesicles, also known as sEVs, by different cells in the spinal cord, can confirm a successful delivery of sEVs. This enables both mechanistic and therapeutic studies of intrathecally administered sEVs from various sources in roden models. This protocol can be used to visualize sEVs uptake by cells in a mild spinal cord, and can help evaluate the role of sEVs and its cargo molecules in spinal disorders, pain and inflammation.
This procedure will be demonstrated by me, along with and Xuan Luo a nd Zhucheng Lin, the two graduate students in Dr Seena Ajit's lab. Begin by placing 18 millimeter cover slips in the 12 well plate, then plate neuro-2a cells in each well with a total of 1 milliliter of complete DMEM containing 10%FBS and 1%pen strep. When the cell count fluency has reached 80 to 90%change the medium to DMEM exosome depleted medium in each well add one, 5 or 10 micrograms of labeled sEVs.
For 1, 4 and 24 hours. For dose and time dependent uptake to the rest of the wells at an equal volume of dye control. Collect the brains of postnatal pups in a 60 millimeter, Petri dish containing ice cold Hanks balanced salt solution supplemented with 10 millimolar hepes, dissect both the cortical lobes and remove the meninges.
Then mince the tissues with a sterilized blade. Transfer the tissues into a 15 milliliter conical tube containing Pepane or deoxyribonucleus one dissociation buffer and incubate for 20 minutes at 37 degrees Celsius, swirling the tube every 5 minutes, aspirate the supernatant and add 5 milliliters of complete DMEM to the tube. To inactivate the enzyme activity, try to rate carefully to dissociate the tissues with a 5 milliliter glass serological pipette, and a flame polished pasture pipette.
Pass the cell suspension through a 40 micrometer cell strainer, and then centrifuge the cells at 250 times G for 5 minutes at 4 degrees Celsius, aspirate the medium and see the cells in 10 milliliters of complete DMEM in a 75 square centimeter flask. After 4 hours of plating, replace the supernate and medium with 15 milliliters of fresh DMEM medium. After 14 days in vitro to detach the microglia and oligodendrocytes, transfer the flask to an orbital shaker at 320 RPM for 6 hours.
Use 5 milliliters of the cell dissociation enzyme for 10 minutes at 37 degrees Celsius. To tripsinize, the remaining astrocytes, to inactivate the enzymatic action, add a 5 milliliters of complete DMEM and then pellet the cells at 250 times G for 5 minutes at 4 degrees Celsius. Resuspend the cells in complete DMEM and see the cells on the 12 millimeter number 1.5 cover slips in a 24 well plate.
Using PBS rinse the cells 3 times and then fix them with 4%power formaldehyde or PFA in PBS for 10 minutes at room temperature. Wash the fixed cells 3 times for 5 minutes with PBS and permeabilize them using 0.1%Triton X 100 in PBS for 10 to 15 minutes. Then repeat the washes with PBS.
Use 5%normal goat serum or NGS in PBS for 1 hour at room temperature to block the cells, incubate the neuro-2a cells with map two-way antibodies and the primary astrocytes with GFAP antibodies at a 1 to 500 ratio in fresh 5%NGS or PBS overnight at 4 degrees Celsius with gentle shaking. After washing the cells 3 times in PBS, add the fluorophore conjugated secondary antibodies in 5%NGS. Incubate for 2 hours at room temperature protected from light on a rocker.
Repeat the washing with PBS and incubate the cell with 1 microgram per milliliter of DAPI for 10 minutes at room temperature. Once again, wash the cells 3 times with PBS. Using an anti fade mounting medium, mount the cover slips on number 1 slides, let them dry overnight in the dark and store the prepared glass slides at 4 degrees Celsius until imaging on a confocal microscope.
Dissect the spinal cord and fix it in 4%PFA in PBS at 4 degrees Celsius for 24 hours. Prior protect the tissues in 30%sucrose in PBS at 4 degrees Celsius for 24 hours or until the tissues sink and then store the tissues at 4 degrees Celsius until immunohistochemistry, imbed the L4 L5 spinal cord in OCT compound, then freeze it on dry ice until it is completely solidified. Use a cryostat to section the tissues at 30 micrometres and the collect the sections in a 24 well plate containing PBS.
Wash the sections 3 times for 5 minutes each with 0.3%Triton in PBS, Block the non-specific binding sites using 5%MGS in 0.3%Triton PBS for 2 hours at room temperature. Incubate the sections overnight at 4 degrees Celsius on a shaker. Wash the sections 3 times for 5 minutes with 0.3%Triton in Pbs.
Then add the appropriate concentration of the secondary antibodies. According to the text manuscript, incubate at room temperature for two hours, wash the sections using only PBS and incubate them in 1 microgram per milliliter of DAPI for 10 minutes at room temperature. Then repeat the PBS washes under a light microscope, mount the sections on a clear adhesive slide with a fine paint brush.
Wet the cover slip with the mounting medium and cure overnight in the dark at room temperature, acquire images under a confocal microscope with the respective lasers, The average mean size of RA 2 6 4 0.7 derived sEVs was 140 nanometers and the peak particle size was 121.8 nanometers. With the most detectable particles falling within the size range of exosomes or sEVS. At 50 to 150 nanometers, western blotting of sEVs, Cell Lysate and exodepleted Media, demonstrated that sEV derived protein samples contained the sEV marker proteins Alix CD81 and GAPDH.
The cell lysate was enriched with endoplasmic reticulum, resident protein Calnexin thus Calnexin served as a negative marker for cellular contamination as it was absent in the sEVs. It was observed that the uptake of sEVs occurred at 1 hour and for the 1, 5 and 10 microgram sEVs, post incubation fluorescence could be detected at 4 hours, for 4, 5, and 10 micrograms of sEVs. The uptake of PKH 26 labeled sEVs by primary astrocytes was examined and maximum fluorescence from sEVS uptake in primary cortical astrocytes occurred at 24 hours PKH26-labeled sEVs were taken up and maximum fluorescence was observed at 6 hours, post-injection in neurons astrocytes and microglial cells.
The inclusion of controls, unlabeled sEVs, and dye control it create code to a wind false positive recent signals due to now specific labeling Opry dance of Unbound dye patch coasts. The uptake of seVs can be confirmed by investigating biomolecular cargo transfer to recipe and cells and tissues and determining the changes in expression levels of biomolecules or target genes. Behavior studies can also be performed, to investigate the functional impact of sEV delivery.
