Biomedical Engineering Reference
In-Depth Information
Fig. 1
Automatic identifi cation of cells. In this example, HeLa cells were loaded for 45 min with Alexa-568-
labeled Tf in a recycling experiment, fi xed, stained with Hoechst 33342 and Alexa-488 phalloidin, and imaged
in the corresponding three channels (Nucl, Phal, and Tf). The nuclei channel is used to identify nuclei (
green
outlines
). Together with the phalloidin channel, this allows to delimit individual cells (outlined in
red
), which in
turn is the basis to quantify the Tf signal per cell
2
Materials
2.1 Cell Culture
1. HeLa Kyoto
cells [
12
] cultured in Dulbecco's Modifi ed
Eagle's Medium containing 10 % fetal calf serum, 2 mM gluta-
mine, 100 U/mL penicillin, and 100
ʱ
ʼ
g/mL streptomycin
(Sigma) at 37 °C with 7.5 % CO
2
.
2. Assay medium consisting of Dulbecco's Modifi ed Eagle's
Medium containing 2 mM glutamine and buffered with
20 mM HEPES-NaOH, pH 7.2-7.5 (Invitrogen).
3. 96-well fl at clear bottom black polystyrene TC-treated micro-
plates (Corning Life Sciences).
4. FuGENE HD transfection reagent (Promega) for plasmid
transfection.
5. Lipofectamine RNAiMAX transfection reagent (Invitrogen)
for transfection of siRNA. Dilutions of transfection reagents
and plasmids or siRNA prior to transfection are made with
serum-free Dulbecco's Modifi ed Eagle's Medium.
