Biomedical Engineering Reference
In-Depth Information
(3)
Replace the growth media at least once every three days until the cells
reach about 80-90% confluence.
(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)
Add 5 mL of fresh RPMI-1640 medium neutralized trypsin in the flask.
(6)
Transfer the cell suspension into a centrifuge tube and pellet the cells at
4000 rpm for 5 min.
(7)
Resuspend the cells in fresh RPMI-1640 and subculture when necessary.
(8)
Centrifuge at 4000 rpm for 5 min to precipitate the cells.
(9)
Resuspend in 1 mL 1× DPBS.
(10)
Count the cells.
(11)
Take 10,000 cells and place in a centrifuge tube.
(12)
Precipitate the cells at 4000 rpm for 4-5 min.
(13)
Resuspend in 10-50 µL DPBS.
(14)
Add 10 µL of 1-50 nM QDs and shake gently.
(15)
Incubate at 37 °C with 5% CO
2
in an incubator oven for 5-15 min.
(16)
Precipitate the cells at 4000 rpm for 4-5 min.
(17)
Wash three or more times with 1× DPBS with 0.02% Tween 20 and 1%
BSA to remove excess QDs.
(18)
Resuspend in 1× DPBS.
(19)
Observe under a fluorescence microscope.
Figure 3.6
shows SK-BR3 cells that were stained with red QDs (QSH620)
from Ocean NanoTech. The cells were rapidly growing when harvested and
exposed to 10 nM QDs for 5 min. Nonspecific staining with QDs shows
almost a very even distribution of the QDs all over the cell in contrast with
specific staining. Staining nonspecifically is useful for microscopic examina-
tion of cells.
Part 2. Cells grown on a cover slip inserted in a 6-well plate
Nonspecific staining of cells can also be done on adherent cells. The follow-
ing protocol is for staining cells attached to glass cover slip:
(1)
Insert a cover slip in each well of a 6-well cell culture plate.
(2)
Place 5000 cells in each well and add 1 mL culture media.
(3)
Incubate the cells under manufacturer's recommended conditions (usu-
ally at 37 °C with 5% CO
2
in an incubator oven).
(4)
When the growth reaches 70-80% confluence, remove the growth medium.
(5)
Add 500 µL of 1× DPBS.
(6)
Add 10 µL of 1-50 nM QDs and aspirate gently to mix.
(7)
Incubate at 37 °C with 5% CO
2
in an incubator oven for 3-10 min.
(8)
Wash three or more times with 1× DPBS with 0.02% Tween 20 and 1%
BSA to remove excess QDs.
(9)
Observe under a fluorescence microscope (
Figure 3.7
). The cells appear flu-
orescent and are easier to find and to count. Sophisticated microscope can
exhibit external and internal features of the cells when stained with QDs.
