3.4.3 Protein
Quantification Assay
The following protocol described steps of using a Pierce™ BCA
Protein Assay Kit. The protocol may need slight modifications for
the application in other assays (see Note 17).
1. Pipette 25 μL of the CDNs samples into a 1.5 mL
microcentrifuge tube.
2. Dilute the samples with 25 μL of PBS (2Â dilution), and stored
on ice prior to the assay.
3. Prepare BSA standards, which can be stored at 4
C for up to
2 weeks.
4. Prepare BCA reagent. Mix it well and protect from light.
5. Load 25 μL of the standards and samples into the wells of a
transparent 96-well plate.
6. Add 200 μL of BCA reagent into each well.
7. Incubate at 37
C for 30 min prior to the analysis at 562 nm
using microplate readers.
4 Notes
1. U937 is a suspension cell line. There is no need for enzymatic
treatment (trypsinization) to detach cells from the surface of
cell culture flask during the subculture or passage. Instead, cells
are continuously maintained by diluting the cells with fresh
culture medium.
2. Cells (either suspension or adherent cells) should be grown to
reach 70–80% confluency. It is important to ensure the cells are
not over confluent, as it may lead to changes in expression of
cell surface protein/markers [46] and gene expression
[47, 48]. Furthermore, when the cells deplete all the nutrients
supplemented in the culture medium, they may cease proliferation and enter quiescence (reversible cell cycle arrest), then
eventually become senescent (irreversibly lose cellular
functions).
3. All centrifugations steps should be performed at 4
C. All the
tubes should be weighed before the centrifugation to ensure
the weight balance during the centrifugation.
The cold condition is important to slow down the enzymatic reaction of intracellular proteases, thus preventing any
degradation and proteolysis.
4. The supernatant should be removed from the tube carefully in
one smooth motion to prevent the disturbance of the cell
pellet. The supernatant consists of spent medium, cellular
debris, and dead cells. The presence of dead cells and debris
may negatively affect the health of other viable cells, by causing
Exosome-Mimetics For Drug Delivery
163
Quantification Assay
The following protocol described steps of using a Pierce™ BCA
Protein Assay Kit. The protocol may need slight modifications for
the application in other assays (see Note 17).
1. Pipette 25 μL of the CDNs samples into a 1.5 mL
microcentrifuge tube.
2. Dilute the samples with 25 μL of PBS (2Â dilution), and stored
on ice prior to the assay.
3. Prepare BSA standards, which can be stored at 4
C for up to
2 weeks.
4. Prepare BCA reagent. Mix it well and protect from light.
5. Load 25 μL of the standards and samples into the wells of a
transparent 96-well plate.
6. Add 200 μL of BCA reagent into each well.
7. Incubate at 37
C for 30 min prior to the analysis at 562 nm
using microplate readers.
4 Notes
1. U937 is a suspension cell line. There is no need for enzymatic
treatment (trypsinization) to detach cells from the surface of
cell culture flask during the subculture or passage. Instead, cells
are continuously maintained by diluting the cells with fresh
culture medium.
2. Cells (either suspension or adherent cells) should be grown to
reach 70–80% confluency. It is important to ensure the cells are
not over confluent, as it may lead to changes in expression of
cell surface protein/markers [46] and gene expression
[47, 48]. Furthermore, when the cells deplete all the nutrients
supplemented in the culture medium, they may cease proliferation and enter quiescence (reversible cell cycle arrest), then
eventually become senescent (irreversibly lose cellular
functions).
3. All centrifugations steps should be performed at 4
C. All the
tubes should be weighed before the centrifugation to ensure
the weight balance during the centrifugation.
The cold condition is important to slow down the enzymatic reaction of intracellular proteases, thus preventing any
degradation and proteolysis.
4. The supernatant should be removed from the tube carefully in
one smooth motion to prevent the disturbance of the cell
pellet. The supernatant consists of spent medium, cellular
debris, and dead cells. The presence of dead cells and debris
may negatively affect the health of other viable cells, by causing
Exosome-Mimetics For Drug Delivery
163