Chemistry Reference
In-Depth Information
FLOUROMAG. D.S.L. was the recipient of a postdoctoral fellowship from the EU
FP5 grant QLG2-CT-2000-02278 awarded to T.M.J. and B.R. was supported by a
TALENT fellowship from the Netherlands OSR.
Appendix 6.A: Materials and Methods
6.A.1
Reagents
Biotin
-
EGF, EGF, and streptavidin-conjugated, -pegylatedQdots were obtained from
Invitrogen. PD153035 was purchased from Calbiochem. Live cell labeling was
carried out in Tyrodes buffer with 20mM glucose and 0.1% BSA. PBS is phosphate
buffered saline.
6.A.2
Cell Lines
HeLa cells, expressing 60 000 EGF receptors per cell, A431 cells, an epidermal
carcinoma cell line expressing 2
10
6
EGF receptors per cell, and the same cell line
stably transfected with EGFR
eGFP were maintained in DMEM with 10% fetal calf
-
serum.
6.A.3
Cell Treatments
Cells were typically starved (0.1% FCS) overnight and were treated with 1
M
PD153035 kinase inhibitor for 2 h at 37
C prior to and during diffusion
measurements.
m
6.A.4
QD Conjugation to Epidermal Growth Factor
Monobiotinylated-EGF was coupled to the streptavidin QDs by incubation with
10 nM QD concentrations in PBS containing 1% BSA for at least 30min at 4
C.
Ratios of EGF: QD were usually 3 : 1 or 1 : 1 unless otherwise speci
ed.
6.A.5
Wide-field Microscopy
Wide-
eld detection of QDs was performed with an Andor iXon DV887 emCCD
camera attached to an Olympus IX71 microscope with a 60
1.45 NA oil immersion
or 60
1.2 NA water objective. QDs were excited by a mercury arc lamp at 436 nm
with a bandpass
filter and appropriate QD (20 nm) emission
filters (Chroma
