Generating Dorsal Root Ganglion Explant and Dissociated Cell Culture
내레이션 대본
Transfer each DRG to a dry, glass Petri dish. Under a surgical microscope, clean and trim off excess fibers and connective tissue still attached to the DRG using a blade. The DRG is easily identifiable as a bulgy, transparent structure along the white spinal nerve and blood vessels are often found surrounding the DRG.
After that, place the cleaned to DRG in a new Petri dish containing ice-cold, serum-free media. Dilute the gelatinous protein mixture in ice-cold, serum-free media in a 1-to-1 ratio. Then, plate the DRG ex vivo in the 12-well plates, precoated with 10 or 20 microliters of gelatinous protein mixture, and keep them at 37 degree Celsius for 30 to 60 minutes.
Now, gently add 1.5 to 2 milliliters of serum-free media to the culture system to cover the entire explant and maintain the explants at culturing conditions. Change the DRG growth medium every 72 hours. and let the DRG grow for as long as needed.
This is a critical step since DRG is anchored to the glass plate using the gelatinous protein mix. Therefore, time of polymerization and pipetting skills are critical to avoid floating.
In this procedure, place all the DRG collected in a 1.5-milliliter sterile tube with F12 media containing collagenase IV, and incubate it at 37 degrees Celsius for 45 minutes. Afterward, replace the fresh media containing collagenase IV, and incubate the sample for another 45 minutes. Then, treat the explants with 2 milliliters of F12 media containing 0.025% trypsin at 37 degrees Celsius for 30 minutes.
Immediately after the collagenase IV treatment, incubate them with 2 milliliters of F12 media containing fetal bovine serum at 37 degrees Celsius for 15 minutes. Afterward, wash the explants three times with 2 milliliters of F12 media, and proceed to mechanically dissociate them with a glass pipette, until the media turns cloudy.
Following that, filter the dissociated cell culture through a 0.22-micrometer filter to remove any impurities and excess connective tissue. Then, centrifuge the filtered cell lysate for two minutes. Remove the supernatant and resuspend the cell pellet in 500 microliters of neuro-basal media. Place the dissociated cells onto laminin-coated coverslides at a preferred cell density.
