Functional Genomics Assays to Study CFTR Traffic
and ENaC Function
Joana Almaça, Shehrazade Dahimène, Nicole Appel,
Christian Conrad, Karl Kunzelmann, Rainer Pepperkok,
and Margarida D. Amaral
As several genomes have been sequenced, post-genomic approaches like transcriptomics and proteomics,
identifying gene products differentially expressed in association with a given pathology, have held promise
both of understanding the pathways associated with the respective disease and as a fast track to therapy.
Notwithstanding, these approaches cannot distinguish genes and proteins with mere secondary patho-
logical association from those primarily involved in the basic defect(s). New global strategies and tools
identifying gene products responsible for the basic cellular defect(s) in CF pathophysiology currently
being performed are presented here. These include high-content screens to determine proteins affecting
function and trafficking of CFTR and ENaC.
Key words: Cystic fibrosis, CFTR, secretory traffic, ENaC, high-content screens, siRNA, functional
The information of complete genome sequences and the identifi-
cation and systematic cloning of human cDNAs provide the chal-
lenging opportunity to analyse the complexity of biological pro-
cesses on a large scale. Systematic approaches, such as organelle
proteomics or yeast two-hybrid screening, have attempted to
identify structural and regulatory components of membrane
J. Almaça and S. Dahimène contributed equally to this work
M.D. Amaral, K. Kunzelmann (eds.), Cystic Fibrosis, Methods in Molecular Biology 742,
DOI 10.1007/978-1-61779-120-8_15, © Springer Science+Business Media, LLC 2011
250Almaça et al.
traffic with the goal of reaching a more complete description
of its molecular regulation. However, these techniques have
limitations, not least of which is their lack of demonstrating a
functional involvement of the molecules identified.
Recent advancesin automated
microscopy and image processing allow the application of com-
plete genome knowledge in large-scale screening applications
with so far unmatched functional information at the single-cell
or the sub-cellular level. Indeed, in combination with genome-
wide/high-content (HC) small-interference RNA (siRNA) or
cDNA over-expression strategies, such microscopy-based appli-
cations hold promise to help revealing comprehensively the reg-
ulatory networks underlying several cellular processes, such as
membrane traffic, in intact cells. These techniques, providing
single-cell or even sub-cellular resolution, are thus of supe-
rior quality and far more informative than conventional high-
throughput (HT) plate reader cell-based fluorescence analyses.
For higher efficiency in dealing with HC siRNA/cDNA libraries,
microscopy-based approaches can employ a ‘reverse transfection’
method, by which hundreds/thousands of siRNAs (or cDNAs)
are pre-spotted on glass chambered slides and subsequently over-
laid with cells which are thus locally ‘reverse transfected’ (1). Crit-
ical in this development is, however, demonstration of robustness
and scalability, two essential prerequisites for large-scale HC func-
tional screens to be conducted.
Functional microscopy-based assays in living or fixed cells,
coupled with large-scale genomic tools, have been recently devel-
oped and applied to solve problems in trafficking of membrane
proteins (2–6), similar to that associated with F508del-CFTR (7).
Such platform was demonstrated as suitable to perform genome-
wide siRNA screens addressing the question of which genes are
required for transport of the temperature-sensitive variant of
vesicular stomatitis virus glycoprotein (ts-O45-G) from the ER
to the plasma membrane (3).
microscopy-based screening technology to study traffic and func-
tion of two CF-related membrane proteins, namely the cystic
fibrosis transmembrane conductance regulator (CFTR) and the
sodium (Na+) epithelial channel (ENaC).
The method described here was developed to be used in HC
microscopy-based assays to monitor the traffic of wt-CFTR and
F508del-CFTR in order to identify relevant intervenients in
this process. To this end, two novel CFTR constructs (wt and
F508del) were generated, namely bearing both an N terminus-
fused fluorescent tag (mCherry) and a Flag epitope tag located at
CFTR 4th extracellular loop (8) (Fig. 15.1a). The Flag tag allows
to quantify the CFTR that is exclusively localized at the cell sur-
face by usage of an antibody applied extracellularly without cell
Functional Genomics of CFTR and ENaC251
Fig. 15.1. (A) Scheme of the novel CFTR constructs (wt and F508del) generated, with
both an N-terminus-fused fluorescent tag (mCherry) and a Flag-tag located at CFTR 4th
extracellular loop and example of microscopy images obtained from the stable A549
cells expressing the mCherry–Flag–wt-CFTR (B) or F508del-CFTR (C) constructs under
a Tet-ON promoter, after induction with 1 μg/ml of doxycycline and 10 mM of sodium
butyrate. The images to detect mCherry intracellular signal were obtained using the
661 nm laser (left panels). The images to detect the Flag staining performed without cell
permeabilization to label exclusively protein at the plasma membrane were obtained
using the 633-nm laser (right panels). Images were acquired using confocal microscope
(LSM710, Zeiss). The pinhole opening was 4.5 μm. Scale bar = 10 μm.
permeabilization. On the other hand, usage of the fused mCherry
tag (9) allows for quantification of the total amount of CFTR
protein expressed by each individual cell assessed by the micro-
scope in the screens. Together, these two tags allow to determine
on each individual cell the fraction of expressed CFTR which is
residing in the cell membrane in the trafficking assay. For the
252Almaça et al.
synchronized expression of CFTR and monitoring of its traffic
through the secretory pathway, these two CFTR constructs (wt
and F508del) were used in the establishment of stable human
epithelial respiratory cell lines (A549 cells) under a tetracycline-
inducible (Tet-ON) promoter. A549 cells are adenocarcinomic
human alveolar basal epithelial cells, and they do not express
The presence of both tags (mCherry and Flag) does not
impede trafficking of wt-CFTR to the plasma membrane, as
shown by staining with the anti-flag antibody without cell perme-
abilization (Fig. 15.1b, right panel). Moreover, functional exper-
iments showed that the mCherry–flag–wt-CFTR is still func-
tional. It produced a cAMP-activated whole cell conductance (42
± 3.2 nS; n = 11) that was comparable to that of non-tagged
CFTR (37.3 ± 2.8 nS; n = 9). In contrast, F508del-CFTR is not
detected at the plasma membrane in A549 cells but is retained
in the intracellular compartment, plausibly in the endoplasmic
reticulum (ER), as indicated by the mCherry signal (Fig. 15.1c,
left panel), thus also recapitulating what is widely known for this
mutant in several other cellular systems (7).
The assays and image processing algorithms developed for the
ts-O45-G screen (5) were the basis for the assays developed for
wt-CFTR and F508del-CFTR described hereunder.
Genomics of ENaC
For ENaC, a functional live-cell assay was selected, based on
the activity of ENaC as sodium channel, which uses the FLIPR
membrane potential (FMP) voltage-sensitive fluorescent (blue)
dye in combination with the specific ENaC blocker amiloride
(Fig. 15.2a). The negatively charged FMP dye enters the cells
depending on their membrane voltage. If the cell membrane volt-
age is depolarized (less negative) due to active/open Na+chan-
nels, which allows transport of Na+ions into the cell, more FMP
dye will be taken up by the cell and thus the fluorescence sig-
nal will be enhanced. Upon inhibition of ENaC with the specific
inhibitor amiloride, cells become hyperpolarized (i.e. more neg-
ative) and FMP fluorescence is quenched, since less FMP dye is
moving into the cell (for examples of results, see Fig. 15.4a).
This assay can be applied to automatic microscopy screens of
HC siRNA libraries spotted onto 384-spot chambered slides
The ‘primary’ assays described here, once being applied in the
context of HT screens using HC siRNA/cDNA libraries, gener-
ate ‘hits’, which are genes/proteins affecting the traffic or func-
tion of CFTR and ENaC. These should nevertheless be validated
by independent siRNAs targeting the same gene. Then, such val-
idated ‘primary hits’ can constitute a basis for the development
Functional Genomics of CFTR and ENaC 253
grown in dexa
Plate on 384-
Add FMP + amil
Fig. 15.2. The functional live-cell FMP-based assay for ENaC. (A) The voltage-sensitive FLIPR dye FMP is taken up by
the cell depending on the membrane voltage (top). (B) Schematic overview of the application of the FMP assay to screen
by automatic microscopy HC siRNA libraries present in 384-spot chambered slides (LabTeks). For examples of images of
the FMP assay performed on A549 cells, see Fig. 4.
of more focussed ‘secondary’ assays specifically investigating
particular aspects or specific pathways or the trafficking/function
of (wt and mutant) CFTR and ENaC.
1. siRNA oligonucleotides (Ambion): Lyophilized siRNAs are
dissolved with Milli-Q water to a final concentration of
2. Lipofectamine 2000 (Invitrogen, Cat. no 11668-019).
3. Sucrose (USB, Cat. no. 21938).
4. Gelatin (Sigma-Aldrich, Cat. no. G-9391).
5. Fibronectin, human (Sigma-Aldrich, Cat. no. F0895) (only
required for spotting).
6. Drying pearls, orange – heavy metal free (Fluka, Cat. no.
254Almaça et al.
7. One-well or eight-well LabTek chambered glass slides
(Nalge Nunc International, Cat. no 177372 or 177402,
respectively) or 384-well, low-volume plates (Nalge Nunc
International, Cat. no. 264360).
8. Sterile filters, 0.45 μm (Millipore, Cat. no. SCHVU01RE).
Prepare the following solutions:
1. 0.2% (w/v) gelatin: Weigh 0.2 g gelatin and dissolve it in
100 ml water, heat this solution to 56◦C for 20 min for dis-
solving. Let it cool down before use. For spotting (not for
coating), add 10 μg/ml fibronectin to the 0.2% gelatin solu-
tion (this is the gelatin/fibronectin stock solution). Filter the
solution with 0.45-μm pore filter (see Note 1).
2. Sucrose/OptiMEM solution (0.4 M sucrose): Weigh 1.37 g
of sucrose and dissolve in 10 ml OptiMEM without shaking
(see Note 1).
3. Transfection stock solution for liquid handler: 18 μl Opti-
MEM containing 0.4 M sucrose + 21 μl Lipofectamine
2.1.2. Traffic Assay
1. OptiMEM I + GlutaMAX I (Gibco, Cat. no. 51985-
2. DMEM/F12 supplemented with 10% foetal calf serum
(FCS) and 2 mM glutamine.
3. Cell lines: Lung carcinoma cell line A549 (ATCC, Cat. no.
CCL-185) stably transduced with lentivirus encoding for
mCherry–flag–wt-CFTR or F508del-CFTR under Tet-ON
promoter (generated by ADV Bioscience LLC, Birming-
ham, AL, USA).
4. Doxycycline (Sigma).
5. Sodium butyrate (Sigma).
6. Phosphate buffered solution (PBS).
7. Hoechst dye solution 33342 (Sigma, Cat. no. B2261).
8. Paraformaldehyde (PFA).
9. BSA (Sigma-Aldrich, Cat. no. A9056).
10. Monoclonal anti-flag M2 antibody produced in mouse
(1 mg/ml; Sigma).
11. Cy5-conjugated anti-mouse secondary antibody (Molecu-
2.1.3. Functional Assay
1. OptiMEM I + GlutaMAX I (Gibco, Cat. No. 51985-026).
2. Mix of insulin/transferrin/selenium (Gibco, Cat. no.
3. Dexamethasone (Sigma, D4902).
Functional Genomics of CFTR and ENaC255
4. Human alveolar type 2 epithelial A549 cells (ATCC, Cat.
5. FLIPR membrane potential-sensitive (FMP) kit (Molecular
Probes, Cat. no. R8042).
6. Amiloride hydrochloride (Sigma, A7410).
inactivated foetal calf serum (FCS), 2 mM glutamine,
insulin/transferrin/selenium (1/100) and 100 nM dexam-
2. Ringer solution: 145 mM NaCl, 0.4 mM KH2PO4, 1.6 mM
K2HPO4, 5 mM glucose, 1 mM MgCl2, and 1.3 mM Ca-
gluconate (pH 7.4) (all chemicals are from Sigma).
3. FMP staining stock solution: Dissolve the powder from one
vial of FMP (kit component A) in 10 ml of assay buffer (kit
component B) (see Note 2).
1. Concentrator (MiVac, GeneVac).
2. Automated liquid handling robot (Microlab Star, Hamil-
ton), equipped with 96-channel head and coolable carrier
blocks for multi-well plates.
3. Heraeus Multifuge 3S (Kendro, Cat. No. 75004361).
4. Contact printers, ChipWriter (Compact and Pro; Bio-Rad
5. Temperature-controlled plate.
6. Solid pins (Point Technologies, Cat. no. PTS 600).
7. Gel drying box for storage of printed LabTeks (The Stewart
2.2.2. Traffic Assay
1. Confocal microscope (LSM 710, Zeiss) 63× objective.
2. Scanning microscope (Scan∧R; Olympus Biosystems).
3. Filters: ET HQ TRITC/DsRED filter set (Ex: 545/30, Em:
620/60) to detect mCherry signal and filter set F36-523
(Ex: 628/40, Em: 692/40) to detect Flag staining (Cy5-
conjugated secondary antibody).
4. 10× objective (Olympus, Cat. no UPSLAPO 10×).
1. Scanning microscope (Scan∧R; Olympus Biosystems).
2. Filter ET HQ TRITC/DsRED sputtered filter set (exciter:
ET545/30; emitter: ET620/60; beam splitter: T570lp).
3. 10× objective (Olympus, Cat. No. UPSLAPO 10×).
4. Automated liquid dispenser (developed at EMBL).
2.2.3. Functional Assay
256 Almaça et al.
3.1. Spotting of
Spotting of the siRNA libraries was performed as described before
(10, 11) (see Note 3).
1. Put 3 μl of sucrose solution (0.4 M) in OptiMEM into a
2. Add 1.75 μl Lipofectamine 2000 to the same tube and then
1.75 μl of water, mix thoroughly.
3. Add to the same tube 0.5 μl of siRNA (stock solution, 30
μM). Add 4.5 μl (or 4.0 μl, if you added two siRNAs) of
water, so as to have a final volume of 11.5 μl, mix thoroughly
(see Note 4).
4. Incubate for 20 min at room temperature (20–25◦C) to
allow the lipid–siRNA complexes to form.
5. Add 7.25 μl of the gelatin (± fibronectin) solution and mix
thoroughly (see Note 5).
6. Transfer 18 μl of each transfection mix into each well of
a 384-well plate with the same final desired layout of the
LabTek (see Notes 6 and 7).
7. Dry the LabTek using a concentrator (MiVac, GeneVac) for
1 h at 37◦C.
8. Store the coated LabTeks in a box with drying pearls (see
3.2. Traffic Assay
To identify proteins affecting CFTR trafficking, the strategy used
was to knock down endogenous proteins with siRNAs. As posi-
tive controls for the assay, siRNAs for CFTR and joint (double)
for COPII components (sec23Aa and sec23Ba) were used. Also
‘scrambled’ siRNA was employed as a negative control. Here-
under, is the step-by-step protocol adopted for the knock-down
assay with control siRNAs performed using the reverse transfec-
tion method. For this assay, eight-well LabTeks coated with these
control siRNAs are used.
3.2.1. Cell Seeding onto
Stably inducible (Tet-ON) A549 cells expressing mCherry–flag–
wt-CFTR or F508del-CFTR (see above) are maintained in
DMEM/F12 supplemented with 10% FCS and 2 mM glutamine
is seeded onto eight-well LabTeks pre-coated with siRNAs as
1. Split almost confluent (∼90%) A549 cells 24 h prior to their
seeding onto siRNA pre-coated LabTeks (see Note 9).
2. Trypsinize and count the cells on early log phase.
3. Seed at 5 × 103cells/well onto siRNA pre-coated eight-well
LabTeks (with ‘scrambled’, CFTR, and sec23Aa/sec23Ba
Functional Genomics of CFTR and ENaC 257
4. Incubate the cells for 48 h in a growth medium at 37◦C with
5. Induce CFTR expression by incubating the cells with 1
μg/ml doxycycline and 10 mM sodium butyrate for another
of Cells on Chambered
The protocol below is used to detect CFTR solely present at the
plasma membrane. It is performed on non-fixed cells and uses an
anti-flag antibody to detect the Flag epitope which is only exter-
nally accessible if the protein is at the membrane (see Note 10).
1. Rinse the LabTek three times with cold PBS.
2. Incubate the cells with the mouse anti-flag antibody (1:500)
in PBS supplemented with 1% bovine serum albumin for 1 h
3. Rinse the LabTek three times with cold PBS.
4. Fix the cells with 4% paraformaldehyde for 20 min at room
5. Wash with PBS.
6. Incubate with rabbit anti-mouse Cy5-conjugated secondary
7. Rinse three times with PBS.
8. Incubate the cells with Hoechst dye (1/1000) for 15 min to
stain the nuclei.
9. Wash the cells with PBS.
3.2.3. Image Acquisition
Images Acquired by
Prior to the automatic image acquisition, high-resolution images
(such as those shown in Fig. 15.1b, c) can be acquired with a
confocal microscope (e.g. LSM710, Zeiss) to observe in detail
the results from the assay.
Functional assays by gene downregulation by siRNA were per-
formed on a widefield Scan∧R microscopy system (Olympus),
which allows to automatically acquire images at different posi-
tions of the well.
1. Set up the auto-focus based on the nuclei on the DAPI
2. Choose exposure time and filter sets according to dye; in this
assay DAPI, Cy3 and Cy5 channels that allow visualizing
nuclei, mCherry and Flag staining signals were, respectively,
3. Choose the number of positions in each well to nine images
4. Set up the first position.
5. Start automated data acquisition.
258 Almaça et al.
Figure 15.3a shows examples of images acquired automat-
ically by this approach. Cells treated with CFTR siRNA (mid-
dle row) show a reduced number of cells expressing mCherry, in
comparison to cells transfected with ‘scrambled’ siRNA (top row),
thus indicating that the siRNA transfection is efficient and that
CFTR siRNAs are a good control to monitor siRNA transfection
in these experiments. The double knock-down of COPII (bottom
row) greatly inhibits CFTR trafficking to the plasma membrane as
indicated by the decrease in the Flag signal (but not in the Cherry
signal), in comparison to cells transfected with ‘scrambled’ siRNA
(top row). This shows that these two siRNAs knocking down
COPII constitute a good positive control to abolish wt-CFTR
traffic in this kind of experiment.
This assay can be scaled up to high-throughput screening
microscopy on siRNA arrays to downregulate hundreds of genes.
3.2.4. Image Processing
and Data Analysis
The goal of this assay is to measure the ratio of total CFTR at the
plasma membrane (assessed by the Flag tag) vs total CFTR pro-
tein expressed (assessed by the mCherry tag) in order to identify
proteins affecting CFTR transport. To do so, image processing is
performed using Labview-based script as described below:
1. The images are first segmented in the Cy3 channel to iden-
tify single cells by their respective mCherry signal.
2. After background correction, the intensity of total CFTR
expressed (measured by mCherry signal) and CFTR
detected at the plasma membrane (assessed by a fluores-
cently labelled monoclonal antibody, flag–Cy5) is measured
for each individual cell.
3. The intensity corresponding to the protein that is trans-
ported to the plasma membrane is divided by the intensity
of the total protein in the cytoplasmic area (mCherry).
4. The median of the ratios is calculated per individual image
and then a mean of these ratios is determined for the nine
images of the same well.
Fig. 15.3. (A) Example of A549 cells reverse-transfected with scrambled siRNA (top row), CFTR (middle row) or sec23Aa
and sec23Ba siRNAs (bottom row). Forty-eight hours after reverse transfection, mCherry–flag–wt-CFTR expression was
induced with 1 μg/ml of doxycycline and 10 mM of sodium butyrate for another 18 h. The intracellular localization was
without cell permeabilization (right row) and visualized using Cy5 filter (Ex: 628/40, Em: 692/40). The nuclei (left column)
stained with Hoechst using DAPI filter (Ex: 350/50, Em: 460/50). Images were acquired with automated Scan∧R system
(Olympus). Scale bar = 30 μm. Summary of quantification of two independent experiments normalized to the scrambled
values (18 images for each condition). (B) Bar graphs representing the mean of the ratio of cells expressing mCherry–
flag–wt-CFTR treated with scrambled siRNA, CFTR or sec23Aa/Ba siRNAs as indicated vs total cells present under the
same conditions (given by the stained nuclei). (C) Bar graphs representing the mean of the ratio of Flag Cy5 (total CFTR
expressed at the plasma membrane) vs total CFTR expressed (mCherry).∗P < 0.05 vs scrambled siRNA.
Functional Genomics of CFTR and ENaC259
Proportion of cells expressing
Relative transport of Wt-CFTR
(flag, Cy5 vs mCherry signal)
mCherry Flag ab (cy5 Ab)DAPI
Fig. 15.3. (continued)
260 Almaça et al.
5. Data are presented as means ± SEM and are analysed for
significant differences using a standard Student’s t-test.
Quantification of automatically acquired images (like those in
Fig. 15.3a) by this approach can provide data on the propor-
tion of cells expressing mCherry-CFTR vs the total number of
cells present or the relative transport of wt-CFTR (Fig. 15.3b, c,
3.3. Functional Assay
The microscopy assay described here for ENaC is a functional
assay, based on the activity of ENaC as a sodium channel. This is
a live-cell assay and uses the FLIPR membrane potential (FMP)
voltage-sensitive fluorescent (blue) dye, in combination with the
specific ENaC blocker amiloride (Fig. 15.2a). The assay uses a
human epithelial cell line, human alveolar type 2 epithelial A549
cell line, which expresses ENaC endogenously, being thus appro-
priate to detect variations in amiloride-sensitive currents (Fig.
3.3.1. Cell Seeding onto
1. Culture human alveolar type 2 epithelial A549 cells in
DMEM/F12 supplemented with 10% FCS, glutamine,
insulin/transferrin/selenium and 100 nM dexamethasone
for several passages (at least for 5 days).
2. Trypsinize A549 cells on early log phase and seed them at
1 × 105cells/ml on pre-spotted one-well LabTek cham-
bered cover glass where 384 different human siRNAs had
3. Allow reverse transfection to occur for 48 h in the CO2incu-
bator at 37◦C.
1. Pre-warm the microscope chamber to 37◦C, turn on CO2
and the fluorescence lamp and the other Scan∧R microscope
2. Dilute the FMP staining stock solution five times with
Ringer solution (see Note 11) and add to half of it amiloride
to obtain FMP/Ringer/amiloride (amil, final concentration
30 μM). Incubate both solutions at 37◦C for 10 min.
3. Add this diluted FMP solution (no amil) to the cell array
(chambered slide; LabTek) and incubate in the microscope
chamber (at 37◦C) for 10 min before imaging.
4. Start imaging each spot in the Scan∧R microscope with a
10× objective in the Cy3 channel, with an exposure time of
25 ms and a gradient-based auto-focus, until spot 384th has
been imaged (one image per spot is acquired). This is the
first cycle of acquisition.
5. Replace the FMP solution by new FMP solution contain-
ing 30 μM amiloride, using an automatic liquid dispenser
Functional Genomics of CFTR and ENaC 261
amiloride 30 µM
Amil-sensitive fluorescence ratio
normalized to scrambled
Fig. 15.4. (A) Example of the FMP assay for cells grown on spotted slides with control
siRNAs. Images show A549 cells incubated with FMP and acquired before and after the
addition of amiloride. As a positive control for the inhibition of ENaC transport, an siRNA
an siRNA targeting INCENP was used as a transfection control and polynucleated cells
can be seen (see Note 12). (B) Bar graph representing the mean ± SEM (n = 4) of
normalized amiloride-sensitive fluorescence ratios for cells grown on scrambled, ßCOP–
and ßENaC–siRNA spots. The normalization applied was (spot fluorescence ratio – mean
scrambled fluorescence ratio)/(2 × SD scrambled fluorescence ratio).∗represents a
significant difference when compared to scrambled normalized ratios (unpaired t-test).
262 Almaça et al.
adapted to the microscope stage (if available), or manually
by carefully using an automatic pipette.
6. Three minutes after incubation with amiloride, start image
acquisition of each spot, by the same order as in the first
cycle, avoiding auto-focusing. This is the second cycle of
acquisition (see Note 11).
Figure 15.4a shows an example of the FMP assay for cells
grown on spotted slides with control siRNAs.
Quantify the intensity of FMP fluorescence per cell before and
after the incubation with amiloride. A ratio between these two
values can be used as a measure of the amount of ENaC that
is expressed and active. The images acquired are processed and
quantified using a Labview-based software by performing the
steps indicated below:
1. Identify the correct pair of images per spot (each corre-
sponding exactly to the same cells before and after amiloride
2. After background correction, segment each cell using the
corresponding FMP fluorescence in each image before
amiloride addition and quantification of the signal (a ‘mask’
3. Apply the same ‘mask’ to the second image of each spot
(after amiloride) and quantify the signal.
4. Calculate the amiloride-sensitive fluorescence ratio for each
cell by applying the following formula:
Ratio =Ibefore− Iafter
where Ibefore is the intensity of FMP fluorescence before
amiloride addition and Iafteris the same parameter after amiloride
1. Calculate the median fluorescence ratio per spot taking into
account each cell in every spot.
2. Normalize the median fluorescence ratio of each spot to the
data obtained for the scrambled siRNA spots in the same
Quantification of automatically acquired images (like those
in Fig. 15.4a) by this approach provides normalized ratios of
median fluorescence ratio per siRNA taking into account each cell
in every spot and different spots for the same siRNA in different
LabTeks (Fig. 15.4b).
Functional Genomics of CFTR and ENaC 263
1. Prepare the gelatin and OptiMEM (containing 0.4 M
sucrose) solutions freshly.
2. The FMP stock solution can be diluted with Ringer solu-
tion, and the cells are still efficiently stained. If stored at
–20◦C, it can be used for 1 week.
3. The procedure here described refers to applying 384 spots
onto a LabTek, but it may be adapted to siRNA coating of
384-microplate or eight-well LabTeks.
4. The siRNAs mix diluted in water is instable; the transfer of
the diluted mix into spotting device should be performed
5. To coat an eight-well LabTeks, transfer 18 μl of each trans-
fection mix into 400 μl of water and then add another 400
μl of water and mix thoroughly.
6. To coat an eight-well LabTek, transfer 100 μl of the diluted
mix to each LabTek.
7. The contact printer will start in the first well of the plate
and print the first row of spots in the LabTek and will con-
tinue like this.
8. LabTeks may be kept for several years stored in a box with
drying pearls at room temperature, provided the pearls are
changed regularly (as soon as they turn white).
9. Split almost confluent (∼90%) cell cultures of A549 cells
24 h prior to their seeding on the dried siRNA LabTek to
have an actively growing cell population to facilitate siRNA
10. Detection of CFTR expressed at the plasma membrane is
performed on non-fixed cells because we observed that cell
fixation alters the Flag staining probably by masking Flag
11. The time of incubation with the FMP staining solu-
tion with and without amiloride is critical and should
be the same between different experiments. Longer
incubations with the FMP solution might lead to an
increase in fluorescence, which may reflect changes in
ionic strength due to activity of endogenous ion chan-
nels. The effect of amiloride is reversible and the second
cycle of image acquisition should not take longer than
264 Almaça et al. Download full-text
12. INCENP is an inner centromere protein, a component of
the chromosomal passenger complex (CPC) that acts as a
key regulator of mitosis. The CPC complex has essential
functions at the centromere in ensuring correct chro-
mosome alignment and segregation and is required for
chromatin-induced microtubule stabilization and spindle
assembly. The siRNA targeting INCENP can work as con-
trol for transfection and spot alignment since the polynu-
cleated phenotype can be easily detected.
This work is supported by TargetScreen2 (EU-FP6-LSH-
2005-037365) grant. J.A. is recipient of Ph.D. fellowship
SFRH/BD/29134/2006 (FCT, Portugal). The authors wish to
thank Beate Neumann and Jutta Bulkescher (Advanced Light
Microscopy Core Facility, EMBL) for their expert technical
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