2.4.2 Protein
Quantification
1. CDNs samples.
2. Standard BCA assay kit.
3. PBS: pH 7.4.
4. 25–2000 μg/mL Bovine Serum Albumin (BSA) standards
(dissolved in PBS).
5. Microplate reader with a 562 nm filter.
6. Clear Flat-bottom 96-well plate.
7. Micropipettes.
3 Methods
3.1 Cell Culture
1. Culture U937 cells in complete culture medium and maintain
at 37
C with 5% CO 2 , until the cells reach 70–80% confluency
(see Note 2).
2. Harvest the cells by centrifuging at 4
C (500 Â g for 10 min)
(see Notes 3 and 4).
3. Remove the supernatant (see Note 5).
4. Resuspend the cells in fresh complete culture medium and
collect an aliquot for cell number determination using the
standard cell counting chamber (see Note 6).
5. Pipette the amount containing 2 Â 10
7 cells into a fresh sterile
15 mL centrifuge tube (see Note 7).
6. Collect the cells by centrifugation at 4
C (500 Â g for 10 min)
and discard the supernatant (see Notes 3 and 4).
7. Wash U937 cells with sterile PBS twice by resuspending the cell
pellet with PBS and repeating the centrifugation step to remove
any residual cellular debris, dead cells, and cell culture medium
(see Note 8).
8. Resuspend the cells in 1 mL of sterile PBS with protease
inhibitor cocktail (1:200 dilution) (see Note 9).
9. Use cells immediately, otherwise leave them on ice and
use within 1 h (see Note 4).
3.2 Cell Shearing Via
Centrifugal Force
The centrifugal force was used in the cell disruption process to
shear the U937 cells to produce the CDNs.
1. Pre-cool the microcentrifuge to 4
C (see Notes 3 and 4).
2. Obtain 10 pieces of 8 μm membrane filters (9 mm diameter)
using the hole puncher (see Note 10).
3. Place two pieces of 8 μm membrane into each spin cup (see
Note 10).
Exosome-Mimetics For Drug Delivery
161
Quantification
1. CDNs samples.
2. Standard BCA assay kit.
3. PBS: pH 7.4.
4. 25–2000 μg/mL Bovine Serum Albumin (BSA) standards
(dissolved in PBS).
5. Microplate reader with a 562 nm filter.
6. Clear Flat-bottom 96-well plate.
7. Micropipettes.
3 Methods
3.1 Cell Culture
1. Culture U937 cells in complete culture medium and maintain
at 37
C with 5% CO 2 , until the cells reach 70–80% confluency
(see Note 2).
2. Harvest the cells by centrifuging at 4
C (500 Â g for 10 min)
(see Notes 3 and 4).
3. Remove the supernatant (see Note 5).
4. Resuspend the cells in fresh complete culture medium and
collect an aliquot for cell number determination using the
standard cell counting chamber (see Note 6).
5. Pipette the amount containing 2 Â 10
7 cells into a fresh sterile
15 mL centrifuge tube (see Note 7).
6. Collect the cells by centrifugation at 4
C (500 Â g for 10 min)
and discard the supernatant (see Notes 3 and 4).
7. Wash U937 cells with sterile PBS twice by resuspending the cell
pellet with PBS and repeating the centrifugation step to remove
any residual cellular debris, dead cells, and cell culture medium
(see Note 8).
8. Resuspend the cells in 1 mL of sterile PBS with protease
inhibitor cocktail (1:200 dilution) (see Note 9).
9. Use cells immediately, otherwise leave them on ice and
use within 1 h (see Note 4).
3.2 Cell Shearing Via
Centrifugal Force
The centrifugal force was used in the cell disruption process to
shear the U937 cells to produce the CDNs.
1. Pre-cool the microcentrifuge to 4
C (see Notes 3 and 4).
2. Obtain 10 pieces of 8 μm membrane filters (9 mm diameter)
using the hole puncher (see Note 10).
3. Place two pieces of 8 μm membrane into each spin cup (see
Note 10).
Exosome-Mimetics For Drug Delivery
161