11. Discard the supernatant and wash the cell pellet with PBS,
centrifuge and remove the supernatant.
12. Add 5 mL–10 mL of sterile DC complete media. Count the
cells.
13. Plate the cells at a density of 1 Â 10
6 /mL. Place in a tissue
culture incubator.
3.2 DC
Differentiation
1. After 12 h in culture, replace the media with fresh DC complete
media.
2. On day 3, remove 75% of the media along with nonadherent
cells and replace with fresh DC supplemented media.
3. On day 6, cells are ready to be replated for future experiments
(see Note 1). Replate the cells using the following protocol:
4. Remove media, which should contain some nonadherent DCs,
and leave the plates with 1 mL of media to prevent adherent
cells from drying out.
5. Detach the cells by adding 10 mM EDTA in PBS to each plate
and allow it to sit for 10 min in the cell culture incubator at
37
C.
6. Gently pipette media up and down against the bottom of the
plate to detach non-adherent DCs for several minutes until
EDTA/PBS thickens.
7. Transfer the cell suspension into a 50 mL falcon tube, and
centrifuge cells at 220 Â g for 5 min at room temperature.
Perform this procedure for each plate.
8. Count and plate the cells for NEP.
3.3 NEP of DCs
1. Plate DCs briefly into the apical chamber of the NEP platform
at a density of 5.0 Â 10
4 /cm
2 per well using DC complete
media.
2. Allow the cells to adhere for >12 h before transfection.
3. Replace the media from the apical and basal chambers for PBS
and plasmid solution, respectively. Place the NEP between a
positive (apical chamber) and negative (basal chamber) electrode, and apply a square wave pulsed electric field of 275 V,
35 ms duration pulse, 10 pulses (Fig. 1) using a Bio-Rad Gene
Pulser Xcell power supply (see Note 2). Pulsing conditions may
need to be adjusted depending on the power supply and/or
type of NEP device.
4. Immediately after transfection, replace PBS for DC completed
media without antibiotics.
5. Incubate cells for 24 h before exosome isolation.
82
Silvia Duarte-Sanmiguel et al.
centrifuge and remove the supernatant.
12. Add 5 mL–10 mL of sterile DC complete media. Count the
cells.
13. Plate the cells at a density of 1 Â 10
6 /mL. Place in a tissue
culture incubator.
3.2 DC
Differentiation
1. After 12 h in culture, replace the media with fresh DC complete
media.
2. On day 3, remove 75% of the media along with nonadherent
cells and replace with fresh DC supplemented media.
3. On day 6, cells are ready to be replated for future experiments
(see Note 1). Replate the cells using the following protocol:
4. Remove media, which should contain some nonadherent DCs,
and leave the plates with 1 mL of media to prevent adherent
cells from drying out.
5. Detach the cells by adding 10 mM EDTA in PBS to each plate
and allow it to sit for 10 min in the cell culture incubator at
37
C.
6. Gently pipette media up and down against the bottom of the
plate to detach non-adherent DCs for several minutes until
EDTA/PBS thickens.
7. Transfer the cell suspension into a 50 mL falcon tube, and
centrifuge cells at 220 Â g for 5 min at room temperature.
Perform this procedure for each plate.
8. Count and plate the cells for NEP.
3.3 NEP of DCs
1. Plate DCs briefly into the apical chamber of the NEP platform
at a density of 5.0 Â 10
4 /cm
2 per well using DC complete
media.
2. Allow the cells to adhere for >12 h before transfection.
3. Replace the media from the apical and basal chambers for PBS
and plasmid solution, respectively. Place the NEP between a
positive (apical chamber) and negative (basal chamber) electrode, and apply a square wave pulsed electric field of 275 V,
35 ms duration pulse, 10 pulses (Fig. 1) using a Bio-Rad Gene
Pulser Xcell power supply (see Note 2). Pulsing conditions may
need to be adjusted depending on the power supply and/or
type of NEP device.
4. Immediately after transfection, replace PBS for DC completed
media without antibiotics.
5. Incubate cells for 24 h before exosome isolation.
82
Silvia Duarte-Sanmiguel et al.