TITLE: Preparing EV-depleted media
AUTHORS: Aizea Morales Kastresana, Bryce Killingsworth, Joshua Welsh, Tim Traynor, Jennifer Jones

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes a method to prepare bovine EV-depleted 10% FBS cell culture media by ultracentrifugation of 20% FBS media and subsequent dilution.</div></div>

[STEPS]
?. [Setting up the centrifugation]
Place 80 mL of FBS and one new 500 mL bottle of media in the 37 ˚C water bath and allow FBS to thaw.
?. [Setting up the centrifugation]
Turn on and log into the ultracentrifuge using the account and password specific to the device.
?. [Setting up the centrifugation]
Write your information on any sign-up sheets associated with the device.
?. [Setting up the centrifugation]
While the FBS is thawing inspect the refrigerated rotor to ensure there are no cracks or other structural issues and that all o-ring seals are intact and greased with vacuum grease.
?. [Setting up the centrifugation]
Obtain a set of six sterilized polycarbonate ultracentrifuge tubes with rubbers stoppers and red aluminum caps. Inspect all the tubes to ensure they look clean and there are no cracks. Generally the tubes will crack after 10 uses, due to stress induced by autoclaving.
?. [Setting up the centrifugation]
Sterilize then transport the thawed FBS, media, and a ultracentrifuge tube rack into a sterile hood.
?. [Setting up the centrifugation]
Pipette 52 mL of media into each tube, avoiding all bubbles and drips on the outside of the tubes (any should be quickly wiped up.)
?. [Setting up the centrifugation]
Pipette 13 mL of FBS into each tube, avoiding bubbles and drips on the outside of the tubes.
?. [Setting up the centrifugation]
Carefully inspect each tube to ensure the exact same volume is in each tube as even small differences can lead to severe rotor or centrifuge damage.
?. [Setting up the centrifugation]
Fasten the stoppers and caps onto the tubes very tightly.
?. [Setting up the centrifugation]
Insert the tubes in the rotor and secure the rotor cap tightly, using a metal rod to tighten the top if necessary.
?. [Setting up the centrifugation]
Transport the filled rotor to the centrifuge.
?. [Setting up the centrifugation]
The vacuum button may need to be pressed to release pressure before the door of the centrifuge can be oppened.
?. [Setting up the centrifugation]
Insert the filled rotor into the ultracentrifuge.
?. [Setting up the centrifugation]
Select the rotor with its specific serial number (etched onto the rotor bottom) in the rotor selection menu.
?. [Setting up the centrifugation]
Create a run at 100,000 x g for 18 hours at 4 ˚C and press vacuum.
?. [Setting up the centrifugation]
Stay with the ultracentrifuge until it reaches its final speed and stop the centrifuge if you have even a slight concern about an unusual sound or movement indicating an imbalance. The ultracentrifuge will hum as it reaches speed but not rattle.
?. [Setting up the centrifugation]
After the device has sufficiently held a vaccum, press the green Start or Go button.
?. [Harvesting the media]
Obtain a new bottle of media which can be cold unless you would like to use the EV-depleted media immediatley.
?. [Harvesting the media]
The moment that the rotor is done spinning, carefully remove the rotor and place it on the centrifuge or adjacent table. If more than 10 minutes elapses between when the rotor stops spinning and you are able to harvest the media you should consider re-centrifuging it as the supernatant may not be depleted of particles any longer.Though the EV pellet is not visible you should handle the rotor and tubes with the understanding that there is a fragile pellet of EVs at the bottom of each of the tubes and any significant movement or josteling can result in part of the pellet becoming resuspended in the supernatant before it is harvested.
?. [Harvesting the media]
Use tweezers or other metal instrument to slowly and gently lift each ultracentrifuge tube from its chamber and place onto the metal rack, minimizing movement and bumping.
?. [Harvesting the media]
Inspect all tubes and tube slots to ensure none were cracked and had their sterility compromised.
?. [Harvesting the media]
Sterilize the outside of the tubes, the rack, the media bottles, and a 0.2 µm cell culture 1 L filter bottle and tranfer all to a hood.
?. [Harvesting the media]
In one motion, pipette off 50 ml from the 65 ml of media in each tube and transfer it on top of the filter
?. [Harvesting the media]
Add 300 mL of media to the ultracentrifuged 20% FBS media, as well as any non-EV containing supplements
?. [Harvesting the media]
Filter the media into the bottle and store for later use
?. [Cleaning]
Remove all items from the hood
?. [Cleaning]
Add tap water to each tube and let sit for at least 30 minutes
?. [Cleaning]
Discard the tap water and refill with tap water for another 30 minutes
?. [Cleaning]
Discard the tap water and, using a centrifuge tube brush and 1% Alconox solution, scrub the insides of each tube and cap and stopper
?. [Cleaning]
Rinse the each tube and cap and stopper first with tap water and DI water. Then re-rinse each tube and cap and stopper with a small volume of pure water at least twice.
?. [Cleaning]
Allow to air-dry, covered to prevent dust accumulation.
?. [Cleaning]
Autoclave using the Gravity 2 setting and a sealable autoclave bag with indicator.