Biomedical Engineering Reference
In-Depth Information
(2)
Incubate the cells under manufacturer's recommended conditions (usu-
ally at 37 °C with 5% CO
2
in an incubator oven).
(3)
When the growth reaches 70-80% confluence, harvest the cells.
(4)
Remove the growth medium and wash the cells two times with DPBS fol-
lowed by 1 mL of trypsin to detach the cells from the flask.
(5)
Transfer the cell suspension into a centrifuge tube and pellet the cells at
4000 rpm for 5 min.
(6)
Resuspend in 1 mL 1× DPBS.
(7)
Take 10,000 cells and place in separate centrifuge tubes labeled 1, 2, 3,
and 4.
(8)
Precipitate the cells at 4000 rpm for 4-5 min.
(9)
Resuspend in 10-50 µL DPBS.
(10)
Add 10 µL of 1-50 nM 2°Ab-QD to tube 1/2 and shake gently for
5 min.
(11)
Add 10 µL of 1 µg/mL anti-HER2/neu to tube 3/4 and shake gently for
5 min.
(12)
Precipitate the cells at 4000 rpm for 4-5 min.
(13)
Remove the supernatant and wash twice with DPBS
(14)
Resuspend in 10-50 µL DPBS.
(15)
Add 50 µL of blocking buffer (an example of a blocking buffer may con-
sist of a combination of 1% BSA, 5% PVP, 0.02% Tween 20, and 0.02%
Triton X in PBS or tris buffer, pH 7.0-7.4) to tubes 1 and 3.
(16)
Shake gently for 5-15 min.
(17)
Precipitate the cells at 4000 rpm for 4-5 min.
(18)
Remove the supernatant and wash twice with DPBS.
(19)
Add 10 µL of 1-50 nM 2°Ab-QD to tubes 3/4 and incubate for 5-30 min.
(20)
Precipitate the cells at 4000 rpm for 4-5 min.
(21)
Remove the supernatant and wash twice with DPBS.
(22)
Resuspend in 10-50 µL DPBS.
(23)
Add 50 µL of blocking buffer (an example of a blocking buffer may con-
sist of a combination of 1% BSA, 5% PVP, 0.02% Tween 20, and 0.02%
Triton X in PBS or tris buffer, pH 7.0-7.4) to tube 3.
(24)
Shake gently for 5-15 min.
(25)
Precipitate the cells at 4000 rpm for 4-5 min.
(26)
Remove the supernatant and wash twice with DPBS.
(27)
Place 1-2 µL of the cell suspensions from each tube on a microscope slide.
(28)
Observe tube under a fluorescence microscope.
Microscopic examination of cells is expected to show QDs for specific
adsorption in tube 3 due to the formation of the complete assay consisting
of SK-BR3 + anti-HER2/neu + 2°Ab-QD. A combination of both specific
adsorption and PA is expected in tube 4 because the cells were not blocked
with a blocking buffer. On the other hand, only PA is expected from tubes
1 and 2 because the cells were not exposed to anti-HER2/neu making the assay
incomplete. However, because tube 1 is washed and blocked, it is expected
