TITLE: Bulk EV staining with CFDA-SE (with NAP-5 purification)
AUTHORS: Aizea Morales Kastresana, Bryce Killingsworth, Jennifer Jones

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A protocol for bulk CFSE staining of EVs.</div></div>

[STEPS]
?. [Preparing CFDA-SE dye aliquots]
Let a new vial of solid CFSE-DA dye warm to RT (should take less than 10 minutes.)
?. [Preparing CFDA-SE dye aliquots]
Per the Vybrant™ CFDA SE Cell Tracer Kit instructions, sissolve the solids in the vial in  anhydrous DMSO (based on the quantity of dye in each vial this creates a  solution.)
90 µl
?. [Preparing CFDA-SE dye aliquots]
Dilute the solution to  total volume with the same anhyrous DMSO, creating a  solution.
4500 µl
?. [Preparing CFDA-SE dye aliquots]
Aliquot volumes of choice into tubes and freeze at , under desiccating conditions to prevent dye hydrolysis during storage.
-20 °C
?. [Dying EVs with CFDA-SE ]
Warm one aliquot of CFDA-SE in your hand, or in a   water bath until just thawed.
37 °C
?. [Dying EVs with CFDA-SE ]
Dilute the CFDA-SE with DPBS to a concentration of   , pipetting then vortexing to ensure the DMSO in the aliquot fully mixes with the DPBS.
?. [Dying EVs with CFDA-SE ]
Prepare a mixture of EVs in DPBS, diluting the mixture to  in a 0.5 mL low protein binding tube.
50 µl
?. [Dying EVs with CFDA-SE ]
Cover with foil and float onto a  water bath for 2 hours, inverting and flicking the tube halfway through the incubation
37 °C
?. [Removing free dye with Nap-5 size exclusion column]
Remove top and bottom caps from column and equilibrate with ~  of DPBS. Ensure that the column is flushed continuously and does not dry
10 mL
?. [Removing free dye with Nap-5 size exclusion column]
Mark 2 Eppendorf LoBind 1.5 mL tubes at the 0.5 mL line on the side of the tube with a pen
?. [Removing free dye with Nap-5 size exclusion column]
Vortex and quickly spin down stained EV mixture
?. [Removing free dye with Nap-5 size exclusion column]
The moment after the last drop of buffer elutes from the Nap-5 column, load the entire  volume of EVs on top of the gel bed of the column and immediatly begin collecting eluate in the first marked 1.5 mL tube.
100 µl
?. [Removing free dye with Nap-5 size exclusion column]
The moment after the loaded EV mixture has been absorbed into the Nap-5 gel, gently fill the buffer reservoir at the top of the column to the top, using ~   of DPBS
3 mL
?. [Dying EVs with CFDA-SE ]
Pipette  of  CFDA-SE into the tube with the EV mixture and vortex to mix
50 µl
?. [Removing free dye with Nap-5 size exclusion column]
After  of eluate has filled the first 1.5 mL tube, start collecting eluate in the second 1.5 mL tube
0.5 mL
?. [Removing free dye with Nap-5 size exclusion column]
Upon elution of  into the second 1.5 mL tube, the Nap-5 column can be filled with DPBS and flushed
0.5 mL
?. [Removing free dye with Nap-5 size exclusion column]
The first 1.5 mL tube is considered Fractions 1 and 2 and should consist of void DPBS and the second 1.5 mL tube is considered Fraction 3 and 4 and sholud consist of eluted EVs
?. [Removing free dye with Nap-5 size exclusion column]
The column should be flushed with ~  of DPBS then loaded with  of filtered 20% Ethanol for storage
10 mL
3 mL
?. [Preparing CFDA-SE dye aliquots]
Determine whether or not you need to prepare new aliquots of CFDA-SE dye.