Biomedical Engineering Reference
In-Depth Information
Chemically Defi ned Serum-free Media (Pro293a-CDM)
without the selection antibiotic. Grow the cells for 2 days. The
Pro293a media (spent media) is harvested and prepared for
immunoblotting or sensitivity assays. The medium is passed
through a 0.45
ʼʜ
fi lter for sterilization.
1. To detect the presence of secreted protein in the media, ali-
quots of 1 mL of spent media (
see
Subheading
3.3.3
) are trans-
ferred to 1.5 mL microfuge tubes. Add 200
3.3.4 Confi rming
Secretion by
Immunoblotting
L 100 % (v/v)
trichloroacetic acid (TCA) to each aliquot (
see
Note 32
). Mix
well and incubate the samples on ice for 30 min. Centrifuge
the tubes for 10 min at max speed, 4 °C.
2. Remove the supernatant without disturbing the fl uffy pellet.
Wash the pellet by adding 1 mL cold methanol-acetone 1:9
(v/v) and inverting the tubes several times. Spin the tubes for
5 min at max speed, 4 °C, and remove the supernatant.
3. Wash the pellet two more times with 300
ʼ
L of methanol-
acetone 1:9. Remove the supernatant and dry the pellet by
placing the tubes in a 95 °C heating block for 5 min.
4. For SDS-PAGE, add 50
ʼ
L 2× sample buffer to the pellet and
mix well. Heat the samples on a dry-block for 5 min at 95 °C
(
see
Note 33
). Vortex the sample after heating and briefl y spin
down the samples. The samples are stored on ice, ready to be
loaded on an SDS-PAGE gel.
5. Twenty-fi ve microliter aliquots of the samples are run on an
SDS-PAGE as described in Subheading
3.3.2
,
steps 4-6
, and
transferred onto nitrocellulose membrane and analyzed by
immunoblotting as in Subheading
3.3.2
,
steps 11-17
.
6. The rest of the samples can be run on a second SDS-PAGE gel
and process with the FOCUS FASTsilver™ staining kit to visu-
alize the amount of protein secreted (Fig.
2
).
ʼ
1. About 150 mL of spent media (SM) is collected from the
stable cell lines (
see
Subheading
3.3.3
). An experimental con-
trol of spent media is collected from cells carrying the empty
vector (EV). The media is sterilized by unit fi ltration of
0.45
3.3.5 Preparation
of Medium Carrying
Secreted Proteins
for Assay
M pore size.
2. The sterile spent media is mixed with fresh medium in a ratio
of 1:4 spent medium to fresh medium (refreshed media, RM;
see
Note 34
).
ʼ
1. The 0.5 % base agar is prepared as in Subheading
3.2.2
,
step 1
.
2. Trypsinize the infected MCF10A cells, and determine cell con-
centration using a hemocytometer. For MCF10A cells,
2.5 × 10
4
cells are used per well. For triplicate wells, 7.5 × 10
4
cells are required.
3.3.6 Soft Agar Assay
(Anchorage Independence)
Using Refreshed Media
