Sep 19, 2020
Bulk EV staining with CFDA-SE (with NAP-5 purification)
- Aizea Morales Kastresana1,
- Bryce Killingsworth1,
- Jennifer Jones1
- 1Translational Nanobiology Section, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health
- Translational Nanobiology Section
External link: https://www.nature.com/articles/s41598-017-01731-2
Protocol Citation: Aizea Morales Kastresana, Bryce Killingsworth, Jennifer Jones 2020. Bulk EV staining with CFDA-SE (with NAP-5 purification). protocols.io https://dx.doi.org/10.17504/protocols.io.ber9jd96
Manuscript citation:
Morales-Kastresana, A., Telford, B., Musich, T.A. et al. Labeling Extracellular Vesicles for Nanoscale Flow Cytometry. Sci Rep 7, 1878 (2017). https://doi.org/10.1038/s41598-017-01731-2
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 07, 2020
Last Modified: September 23, 2020
Protocol Integer ID: 35361
Keywords: flow cytometry, EVs, CFSE, bulk staining,
Disclaimer
This protocol summarizes key steps for a specific type of method, which is one of a collection of methods and assays used for EV analysis in the NCI Translational Nanobiology Section at the time of submission of this protocol. Appropriate use of this protocol requires careful, cohesive integration with other methods for EV production, isolation, and characterization.
Abstract
A protocol for bulk CFSE staining of EVs.
Guidelines
- When possible CFDA-SE dye and CFSE-stained EVs should be protected from significant light exposure
- Fresh aliquots of CFDA-SE should be prepared from solid CFDA-SE dye every few months to avoid using dye with diminished dying ability due to hydrolysis (however in our lab older aliquots of dye have been used without reduced staining capacity)
- See this link for useful information about handling CFSE and using it for labeling cells for flow cytometry: https://med.virginia.edu/flow-cytometry-facility/wp-content/uploads/sites/170/2015/10/Protocol-for-CFSE-Labeling.pdf
Materials
MATERIALS
DPBSInvitrogen - Thermo FisherCatalog #14190
DMSO, AnhydrousThermo FisherCatalog #D12345
Vybrant™ CFDA SE Cell Tracer KitThermo FisherCatalog #V12883
illustra NAP-5 Columns GE HealthcareCatalog #17085301
For preparing CFDA-SE aliquots:
- 1 vial of solid dye from Vybrant CFDA-SE Cell Tracer Kit
- Anhydrous DMSO
- Small volume tubes for aliquoting CFDA-SE (0.5 mL or PCR tubes)
- Large volume tube (5 mL snap tube or 15 mL conical) for diluting CFDA-SE
For staining EVs with CFDA-SE:
- 1 aliquot of CFDA-SE
- 1 Nap-5 column
- 1 500 mL bottle of DPBS
- 1 ring stand to hold Nap-5 column
- Eppendorf 0.5 mL and 1.5 mL LoBind tubes
- Preparation of EVs to stain
- Foil or other cover to protect stained EVs from light
- 37 ˚C water bath or heat block
Preparing CFDA-SE dye aliquots
Preparing CFDA-SE dye aliquots
Determine whether or not you need to prepare new aliquots of CFDA-SE dye.
Let a new vial of solid CFSE-DA dye warm to RT (should take less than 10 minutes.)
Per the Vybrant™ CFDA SE Cell Tracer Kit instructions, sissolve the solids in the vial in 90 µL anhydrous DMSO (based on the quantity of dye in each vial this creates a 10 millimolar (mM) solution.)
Dilute the solution to 4500 µL total volume with the same anhyrous DMSO, creating a 200 micromolar (µM) solution.
Aliquot volumes of choice into tubes and freeze at -20 °C , under desiccating conditions to prevent dye hydrolysis during storage.
Dying EVs with CFDA-SE
Dying EVs with CFDA-SE
Warm one 200 micromolar (µM) aliquot of CFDA-SE in your hand, or in a 37 °C water bath until just thawed.
Dilute the CFDA-SE with DPBS to a concentration of 40 micromolar (µM) , pipetting then vortexing to ensure the DMSO in the aliquot fully mixes with the DPBS.
Prepare a mixture of EVs in DPBS, diluting the mixture to 50 µL in a 0.5 mL low protein binding tube.
Pipette 50 µL of 40 micromolar (µM) CFDA-SE into the tube with the EV mixture and vortex to mix
Cover with foil and float onto a 37 °C water bath for 2 hours, inverting and flicking the tube halfway through the incubation
Removing free dye with Nap-5 size exclusion column
Removing free dye with Nap-5 size exclusion column
Remove top and bottom caps from column and equilibrate with ~ 10 mL of DPBS. Ensure that the column is flushed continuously and does not dry
Mark 2 Eppendorf LoBind 1.5 mL tubes at the 0.5 mL line on the side of the tube with a pen
Vortex and quickly spin down stained EV mixture
The moment after the last drop of buffer elutes from the Nap-5 column, load the entire 100 µL 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.
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 ~ 3 mL of DPBS
After 0.5 mL of eluate has filled the first 1.5 mL tube, start collecting eluate in the second 1.5 mL tube
Upon elution of 0.5 mL into the second 1.5 mL tube, the Nap-5 column can be filled with DPBS and flushed
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
The column should be flushed with ~ 10 mL of DPBS then loaded with 3 mL of filtered 20% Ethanol for storage