Biology Reference
In-Depth Information
viewing chamber to hold the coverslip (35 mmAttofluor cell chamber, Invitrogen)
and ultra fine tweezers for mounting coverslips into the viewing chamber.
7.
Substrate to coat glass coverslips for cell adhesion
Examples include poly-
D
-lysine (HEK293 or CHO cells), laminin (neuronal
cells), fibronectin/collagen (smooth muscle cells), or purchase precoated
MatTek dishes.
8.
A dilute solution (20 nM) of rhodamine 6G, fluorescein (pH
7.5), or GFP for
instrument calibration. HPLC-grade water for dilution of dye and for placing on
the 40
>
water immersion objective
9.
Isotonic solution
For washing cells and performing FCS experiments, use phosphate-buffered
saline, Krebs' ringer solution, or phenol red-free minimal essential media
(MEM, CellGro). Add 10 mM HEPES to the solution to keep pH constant
during FCS recording. Avoid substances with fluorescence such as phenol.
10.
Ethanol and lens cleaning solution
Clean the bottom of the coverslip or MatTek dish immediately prior to the FCS
experiment. Use ethanol on a Kimwipe to clean glass and wipe dry, and then use
a lens cleaning solution and wipe dry.
10.2
METHODS
10.2.1
Sample preparation
10.2.1.1
Choice of cell type
HEK293 and CHO cells are cell types that work nicely for FCS experiments. Primary
cell cultures, such as neurons or epithelial cells, endogenously expressing the recep-
tor of interest may be used. However, this requires a suitable fluorescent tag capable
of labeling native receptors in an exact 1:1 stoichiometry. Membrane stability is im-
portant as movement of the membrane within the observation volume during an FCS
recording will directly impact the diffusion time and molecular brightness.
10.2.1.2
Choice of fluorescent tag
The ideal fluorescent tag has a large quantum yield (brightness), has a good photo-
stability, and is monomeric. The higher the quantum yield, the better the signal to
noise ratio. Photostability is important since photobleaching will decrease fluores-
cence intensity, producing an artificially fast diffusion time as bleaching will mimic
the disappearance of the fluorescence signal from the observation volume. GFP var-
iants, such as eGFP and eYFP, and the newer variants are suitable for FCS. Others,
such as CFP, mCherry, and dsRed, have low quantum yield and have the potential to
form aggregates (dsRed).
10.2.1.3
Labeling the receptor
Plasmids containing GFP variants are useful for attaching fluorescent labels to the
C-terminus of the receptor of interest. This is a commonly employed method to en-
sure an exact 1:1 labeling of the receptor with the fluorescent probe, which is critical
