Biomedical Engineering Reference
In-Depth Information
period of incubation after transfection (typically 3-5 days).
Evaluate the transfection effi ciency (such as the percentage of
cells transfected with fl uorescent siRNA, percent of cells with
fl uorescent reporter knocked down, or percent of cells with
expected phenotype), cell toxicity (by staining and counting
cells or a metabolic or redox assay kit such as CellTiter-Glo or
WST-1/MTT), and dynamic range of the assay—good separa-
tion between positive and negative controls (
see
Note 8
).
3. Once conditions are optimized, validate the complete protocol
on at least one whole plate, including controls in the assigned
wells, to assess the signal-to-noise ratio, as well as test for any
potential artifacts introduced during the experimental proce-
dure (
see
Note 9
).
4. Acquire images of the plate using the same automated HCS
microscope to be used for the screen. Decide on the objective
and number of fi elds of view to be taken per well, to obtain a
suffi cient number of cells per knockdown condition. Optimize
acquisition settings to obtain a good dynamic range, yet allow
detection of perturbations, e.g., if an increase in signal is
expected in target hit genes, ensure that the acquired images
for the controls are not too near saturation.
5. Test the image analysis software to be used, which can be chosen
from either open source (e.g., CellProfi ler [
30
]) or commercial
sources (e.g., Molecular Devices) (
see
Note 10
). Follow this
simple image analysis pipeline: measure the total lectin intensity
of the fi eld of view, count the number of cells using nuclei, and
divide to get lectin intensity per cell. Software such as
ScreenSifter [
31
], cellHTS [
32
], or MS Excel can be employed
to visualize readouts by plate. Image analysis software them-
selves may also include the capability to display plate heatmaps
(e.g., MetaXpress, CellProfi ler).
6. (Optional but recommended) To optimize handling proce-
dures, perform a “dry” run with substitute reagents (e.g., PBS)
with the same number of plates planned for the actual screen.
We recommend processing at most about 8-10 plates per batch.
3.2 Cell Seeding,
Fixing, and Staining
1. Thaw previously prepared siRNA plates in the cell culture
hood for 30 min.
2. Spin the plates down at 200 ×
g
for 1-2 min, before removing
the plate seals.
3. Pipette control siRNAs into the desired wells (
see
Note 11
).
4. Dilute HiPerFect transfection reagent into Opti-MEM and
incubate for 5 min (
see
Note 12
). Use a liquid dispenser such
as the Multidrop Combi or a semiautomated pipette to dis-
pense the transfection reagent uniformly into the plate wells.
5. Incubate the plates for 20 min at 200 rpm on an orbital shaker
to allow transfection complex formation.
