Biology Reference
In-Depth Information
This allows the use of objective lenses with high numerical apertures for maximum
light collection.
Native or h-coelenterazine (supplied by Molecular Probes, Invitrogen, US or
Interchim, France; 1-5 mM stock solution prepared in 100% ethanol).
b. Methods
i. Preparation of cells that are transiently transfected with GFP-
apoaequorin. Healthy cell monolayers can be transfected when they are approxi-
mately 50-75% confluent and imaged within 24-48 h following transfection. Wash
cells 1
and resuspend with serum-free medium (with no antibiotic/antimycotic)
and then place cells back into the 37
C/5% CO
2
incubator. For transfecting a 35-
mm dish, prepare a microcentrifuge tube containing 150
m
l of serum-free media.
Add 4.5
m
l of transfection reagent (this amount may be increased according to the
cell type or reagent used). Vortex the tube and then add 1.5
m
l of plasmid DNA
(1 mg/ml). Mix by flicking the tube and leave the tube at room temperature for
approximately 30-45 min to allow the formation of a complex. Following incuba-
tion, remove the cells from the incubator and add 100
m
l of the mix drop-wise and
gently swirl the dish before placing back into the incubator. Although optional,
healthy cells can be more easily maintained if the medium is changed after 6 h with
fresh medium containing fetal calf serum (FCS). After 24-48 h, replace the normal
growth medium with a serum-/phenol red-free medium (used for imaging), and
incubate the cells for 1 h with 5
m
M coelenterazine (native or h-according to the
type of study). Single cells expressing high levels of GFP-aequorin can be selected
by their GFP fluorescence, and used for bioluminescence Ca
2
รพ
imaging studies.
ii. Preparation of stable cell lines expressing GFP-apoaequorin. Follow the
protocol for preparing cells for transient transfection (see previous section). At
48 h after transient transfection, start the selection process by adding a selective
medium containing the appropriate antibiotic (e.g., Geneticin, or Puromycin). The
antibiotic concentration used (starting at an upper concentration of 1 mg/ml)
needs to be optimized for di
erent cell types. The medium should be changed
with fresh selective medium every 2-3 days over a period of several weeks. During
this time, the concentration of antibiotic may be gradually decreased. Isolation of
GFP-positive clones into 96-well plates can then be facilitated using flow cytome-
try. In our experience, approximately 10% of the clones survive and proliferate
after a first round of FACS sorting. Since stable transfection is a random integra-
tion event and a large amount of variability is expected, the clones should be
selected based on the level of GFP intensity and homogeneity as well as cellular
morphology, and where instrumentation is available (e.g., Nikon's Biostation or
the Incucyte from Essen Instruments), clones can also be selected based on growth
curves.
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