Target Discovery and Validation Reviews and Protocols

Transfected Cell Arrays 159
2. The dwell time the spotter pins remain in the source plate is set to 0.5 s.
3. The dwell time the spotter pins remain on the Lab-Tek chamber is set to 0.1 s.
4. The washing procedures between the spotting of distinct samples are set as follows:
a. Wash container: 10 s.
b. Sonication container: 10 s.
c. Vacuum drying: 10 s.
3.3. Plating of Cells on Lab-Tek Dishes With Dried Transfection Solution
The density of the cells plated on the dried spots of a Lab-Tek chamber is
always a compromise between the improved statistics that can be achieved with
high cell densities and the quality of the microscopic analyses that is best at low
cell densities.
1. Typically, we plate 1.25 × 10 5 actively growing HeLa, MCF7, COS7L, or
HEK293 cells in 2.5 mL of culture medium (Dulbecco’s modified Eagle’s
medium containing 10% heat-inactivated fetal calf serum, 2 mM glutamine,
100 U/ml penicillin, and 100 µg/mL streptomycin) on the dried spots of one
Lab-Tek culture dish. This process results in approx 100 to 200 cells residing
on one spot.
2. The incubation time (at 37°C and 5% CO 2 ) for the successful expression of
plasmid cDNAs varies between 12 and 24 h.
3. The incubation time for RNA interference (RNAi) experiments varies between 20
to 50 h and strongly depends on the stability of the proteins targeted by the
siRNAs spotted. For long-term experiments lasting several days, e.g., RNAi experiments
targeting stable proteins, the cell density needs to be lowered compared with
the density typically used (see step 1), because cells may stop growing because of
contact inhibition at later time-points of the experiment (e.g., 60 h), which makes
experiments addressing cell cycle- or signal transduction-related questions difficult
to interpret.
3.4. Preparation of Samples for Functional Analysis
For functional analysis involving high-content screening microscopy, we frequently
use immunofluorescence (6) to monitor molecule-specific morphological
and biochemical parameters. The immunostaining procedure in Lab-Tek chambered
glass cover slips is very effective and cheap because it can be performed
with the same antibody for hundreds of target genes in parallel.
1. For the staining of Lab-Tek chambers, we apply 250 µL of the correspondingantibody
by carefully distributing the fluid over the spotted area.
2. Incubate for 10 min with the lid closed to avoid rapid evaporation.
3. Wash two times with 2 mL of phosphate-buffered saline (PBS) (30 min each).
4. Stain cell nuclei with 0.2 µg/mL Hoechst B2261 (Sigma). The strong nuclear
staining achieved in this way facilitates automated focusing during image acquisition
(3) (see Subheading 3.5.).