Biomedical Engineering Reference
In-Depth Information
3.3 Coating Plates
with Poly-d - Lysine
1. Pipet 0.5 mL of poly-
D
-lysine solution into each of the wells of
the 48-well plates. Incubate for 5 min at room temperature.
2. Remove poly-
D
-lysine from wells by pipetting into a fresh new
50 mL tube. This recycled poly-
D
-lysine can be stored for reuse in
a subsequent experiment. Wash the wells twice with PBS. Plates
can then be used immediately or stored at −20 °C for later use.
1. Aspirate media from the cells stably expressing the
-lac con-
struct. Wash cells with 5 mL PBS. Then add 1 mL of 0.05 %
trypsin/EDTA and incubate for 1-5 min at RT.
2. Stop trypsin reaction by adding 5 mL of DMEM/FBS media.
Gently resuspend cells by pipetting up and down with a sero-
logical pipette.
3. Count cells using a hemocytometer and dilute to
200,000 cells/mL. Add 0.5 mL/well for 48-well plates
(100,000 cells/well) (
see
Notes 2
and
3
). Seed cells so that
there are three identical wells for each experimental condition
(triplicates).
4. Incubate for a minimum of 6 h at 37 °C to allow cells to
adhere.
5. Apply drug treatment or other desired experimental intervention.
ʲ
3.4 Plating Cells
1. Dissolve 100
L nitrocefi n stock solution (10 mM) aliquot in
9.9 mL of PBS (1:100). This gives a 1× solution (100
ʼ
3.5
β
-Lac Assay
M).
2. Aspirate media from 48-well plate and gently wash cells with
0.5 mL PBS (
see
Note 5
). Repeat the washing once.
3. Add 200
ʼ
L of nitrocefi n solution per well.
4. Immediately read absorbance at 486 nm in each well using a
plate reader. Reading should be carried out every minute for a
period of 30 min.
5. At the end of the assay, a color change should be clearly evi-
dent (Fig.
1
) (
see
Notes 6
and
7
).
6. Plot the absorbance over time in Graphpad Prism or other sta-
tistical analysis software. Perform a linear regression to calcu-
late the slope of the curve (Fig.
2
). The slope should be linear
with an
r
2
value of at least 0.95. Changes in surface expression
are calculated relative to untreated control wells, the slope of
which is set at 100 %.
ʼ
4
Notes
1. Although the
-lac assay can be conducted using a transient
transfection population, our studies show that for reproduc-
ible measurements of internalization studies it is best to use
ʲ
