1. INTRODUCTION
• Concept
2. CELL CULTURE AND TREATMENTS
3. FIXING AND STAINING
4. REAGENTS
• Plates
• Coating
• Fixatives
• Antibodies
• Buffers
• Substrates
5. ASSAY VALIDATION
• Experimental factors
• Results from Response Surface Experiment
• Variables evaluated
• Optimal Conditions Results
6. APPENDIX 1. IMMUNOBLOT VS. CYTOBLOT ASSAYS FOR ENZYME INHIBITOR POTENCIES
7. APPENDIX 2. LINEARITY AND SENSITIVITY OF CELL-BASED WESTERN ASSAYS

In the past several years there has seen an explosion in the availability of commercial sources of antibodies to cell signaling molecules. In addition, antibodies that selectively recognize post-translationally modified proteins (e.g., phosphorylated, acetylated) have also become available. In many cases, these antibodies are very high quality as determined by strength of signal and specificity on immunoblots. These same high affinity, specific antibodies can also be used to detect antigens in fixed cells by immunofluorescence. The spatial and temporal information derived from these studies can be extremely valuable in delineating the function of post-translational modifications. For example, phosphorylation can be a trigger for a change in subcellular localization and consequently, a change in protein function.

Unfortunately, for the purposes of high throughput drug discovery, both immunoblots and immunocytofluorescence have severe restrictions. Immunoblots are not readily adaptable to 96-well plate formatted experimental designs. Although chemilumescent detection systems have made immunoblots extremely sensitive, quantitation is limited by the small linear range inherent in exposed film. More quantitative methods using newer instrumentation (e.g., phosphoroimagers) alleviate some of these difficulties; however, the laborious procedures for preparing cell lysates, determining protein concentrations, loading gels and blotting remain. Immunocytofluorescence with conventional microscopy is also tedious, relatively insensitive and non-quantitative. In order to take advantage of the high quality antibodies available for studying cell-signaling pathways, we have developed procedures for combining the best properties of immunocytofluorescence and immunoblots. This cell-based western (also referred to as cell-based ELISA or C-ELISA) has proved to be extremely useful for medium throughput screens of kinase inhibitors and, in conjunction with biochemical assay data, has become an important SAR driving force.

The key ingredient for a successful cell-based Western assay is a high quality antibody. Single band antibody specificity must first be established by conventional immunoblot procedures. Once a suitable antibody is identified, it can be used to stain and quantitate the levels of antigen in cells in individual wells of a 96-well plate. Cells are plated, treated according to experimental requirements and fixed directly in the wells, similar to an immunocytofluorescence experiment. After fixing, individual wells go through the same series of steps used for a conventional immunoblot, including blocking, incubation with first antibody, washing, incubation with second antibody, addition of chemilumescent substrates and development. Finally, results are read in a luminescence plate reader. Other readouts including fluorescence can be used in this assay format. In our experience, this procedure provides rapid, quantitative analysis of cellular antigens. In general, the correlation between immunoblot and C-ELISA assays is quite good (Appendix 1). The wide linear range of the C-ELISA allows for quantifying >5-fold changes in cellular protein levels in response to stimulant (Appendix 2). The versatility is only as limited as the availability of high quality antibodies to the target proteins.

Cell-based ELISA/Western Schematic

1. Seed cells in a 96-well plate at near confluence 1 day prior to the experiment in 100 ul of growth medium. Optimal seeding density should be experimentally determined (see below).
2. For poorly adherent cells, it is useful to plate cells in wells coated with poly-D-lysine or other extracellular matrix components.
3. To avoid chemiluminescent signal spillover between wells, cells should be plated on white plates. Clear bottoms are convenient because cells can still be viewed under the microscope. However, there is a tendency towards “edge effects” with these plates. Opaque bottom white plates can minimize this problem. Alternatively, outside rows and columns can be excluded from the experimental format. When a fluorescent readout is being used a black plate should be used.
4. For growth factor-stimulated responses, the signal window is often increased if cells are serum starved prior to the experiment. Starvation can be for 2-4 h or overnight depending on the cell type and should be optimized using the experimental design templates (see example below).
5. Preincubate with compounds ∼1-2hr before adding the activating stimulus. Compounds should be added such that the final DMSO concentration is ≤0.25%. The compounds are diluted into medium containing serum or if in serum free conditions add BSA to a final concentration of 1%. Dilute the stocks serially to a final concentration of 10X. You will be adding 10 ul to a total of 100 ul to give a final concentration of the compound of 1X.
6. Prepare activators as 10x stocks in medium + 1.0% BSA. This is usually enough BSA to prevent peptides and small molecules from sticking to the sides of the test tubes. Return cells to incubator for the appropriate time.
7. Stimulate the cells with the reagent that is known to specifically produce the desired response. The concentration of the stimulant and treatment times will be determined by experimental design.

1. *Stop the reactions by inverting plate and dumping media into appropriate waste container. Tap gently on absorbent paper several times to remove residual liquid. For suspension cells, see note 1 below.
2. Add fixative (100 ul/well). The standard fixative is 3.7% formaldehyde diluted from commercially available 37% stock solution into PBS. Alternatively, non-toxic fixatives have recently become available (See reagents below). Usually a 10-minute incubation at room temperature is sufficient, but this may vary with cell type. Other types of fixatives include glutaraldehye, methanol, etc.
3. Invert plate and dump fixative into appropriate waste container. Tap gently on absorbent paper several times to remove residual liquid.
4. Rinse 3 times with PBS + 0.1% Triton X-100 to permeabilize the cells. Incubate 5 min each time.
5. Invert, dump and blot. Add blocking buffer for one hour to block non-specific sites. We have normally used 10%FBS in PBS. At this point, plates can be stored at 4° overnight (for several days).
6. Invert, dump and blot. Add first antibody (50-100 ul/well) diluted in Blocking Buffer or PBST + 1%BSA. Incubate 2 hr at room temperature or overnight at 4°. As a guideline, use first antibody concentrations the same as or 2-fold more concentrated than the optimal immunoblot dilution. The optimal concentration will be determined by experimental design. Incubate the cells with the primary antibody for one hour at room T or overnight at 4°.
7. Invert, dump and blot. Wash 3 times with 100 ul/well PBS + 0.1% Triton (Wash Buffer). One-minute incubation is sufficient at this step.
8. *Invert, dumb and blot. Add 100 ul/well of horseradish peroxidase coupled second antibody of appropriate species specificity. In our hands, a 1:1000 dilution of Amersham Pharmacia antibodies in Blocking Buffer is optimal. Incubate 1hr at room temperature. See note 8 for alternative second antibodies.
9. Towards the end of the 1hr incubation, prepare the commercially available chemiluminescent substrate solution. Mix equal volumes of the luminol/enhancer and stable peroxide reagents. Protect from light while solutions equilibrate to room temperature.
10. *Remove second antibody (invert, dump and blot). Wash wells 2 times with 100 ul Wash Buffer and then 3 times with 100 ul PBS. It is important to completely remove the Triton, which interferes with the peroxidase activity.
11. * If the experiment involves more than 1 plate, process only 1 plate at a time. Leave others in the final PBS wash. Starting with the first plate, invert, dump and blot. Add 100 ul/well of substrate solution. Wait 1 min. Read luminescence (relative light units, RLU) on standard plate reader at 0.1 second (or longer)/well.
12. While first plate is being scanned, develop second plate, etc.

1*. For suspension cells, aliquot cells into wells of a poly-D-lysine-coated plate. We normally spin the cells in the plate for 5 minutes at 1000-1500RPM and wait one hour to allow the cells to rest prior to compound addition or stimulation. The alternative method would be to proceed with the reaction after plating the cells and then stopping the reaction by spinning cells at 1000-rpm for5 min. Invert, dump, and blot. Proceed with fixation as for adherent cells. At this point the cells will be well stuck to the plate.
8*. Fluorescent secondary antibodies can also be used with detection on:
• High information/content laser scanning detection instruments are commercially available and have been used extensively for these applications.
• The use of infrared fluorescent labels can be used in the cELISA assays by using commercially available infrared imaging systems.
10*. The development time significantly influences the luminescence. Therefore, it is not practical to compare the absolute values of RLU between plates. Each plate must contain the appropriate controls, such as min and max or standard curve.
11*. One minute is a convenient reaction time. Although the absolute values of the RLU increase with time, the relative values are consistent for 5 to 10 minutes.

• White plates for chemiluminescent readout
• Black plates for fluorescent readout
• Clear plates if reading colorimetric

• Poly-D-lysine
• Collagen
• Gelatin
• Fibronectin

• 3.7% Formaldehyde (Sigma catalog #F1635 37% solution) in PBS
• Commercially available non toxic fixative reagents
• Methanol

• Primary antibody specific for protein of interest
• Secondary antibody:
• For chemiluminescence: anti-rabbit or anti-mouse IgG HRP conjugate
• For fluorescence detection using the high content imaging technology use: anti- mouse or anti-rabbit IgG Alexa 488
• For fluorescence detection using the the infrared use the reagents labeled with IR tags

• Phosphate buffered saline
• Wash buffer: PBST (PBS with 0.1% Triton X-100)
• Antibody dilution and blocking buffer: PBS with 0.5% BSA and 0.1% Triton X-100

• Colorimetric: TMB
• Chemiluminescent: Commercially available chemiluminescent reagents

1. Check all pipettors to ensure that each channel is precise and accurate.
2. Determine signal window. For statistical analysis, it is important to verify that well-to-well variation is minimal and that the signal window (i.e., difference between min and max values) is large enough to yield reproducible analyses. Perform the C-ELISA using the template provided below.

Plate 1

Row	C1	C2	C3	C4	C5	C6	C7	C8	C9	C10	C11	C12
1	H	M	L	H	M	L	H	M	L	H	M	L
2	H	M	L	H	M	L	H	M	L	H	M	L
3	H	M	L	H	M	L	H	M	L	H	M	L
4	H	M	L	H	M	L	H	M	L	H	M	L
5	H	M	L	H	M	L	H	M	L	H	M	L
6	H	M	L	H	M	L	H	M	L	H	M	L
7	H	M	L	H	M	L	H	M	L	H	M	L
8	H	M	L	H	M	L	H	M	L	H	M	L

Plate 2

Row	C1	C2	C3	C4	C5	C6	C7	C8	C9	C10	C11	C12
1	L	H	M	L	H	M	L	H	M	L	H	M
2	L	H	M	L	H	M	L	H	M	L	H	M
3	L	H	M	L	H	M	L	H	M	L	H	M
4	L	H	M	L	H	M	L	H	M	L	H	M
5	L	H	M	L	H	M	L	H	M	L	H	M
6	L	H	M	L	H	M	L	H	M	L	H	M
7	L	H	M	L	H	M	L	H	M	L	H	M
8	L	H	M	L	H	M	L	H	M	L	H	M

Plate 3

Row	C1	C2	C3	C4	C5	C6	C7	C8	C9	C10	C11	C12
1	M	L	H	M	L	H	M	L	H	M	L	H
2	M	L	H	M	L	H	M	L	H	M	L	H
3	M	L	H	M	L	H	M	L	H	M	L	H
4	M	L	H	M	L	H	M	L	H	M	L	H
5	M	L	H	M	L	H	M	L	H	M	L	H
6	M	L	H	M	L	H	M	L	H	M	L	H
7	M	L	H	M	L	H	M	L	H	M	L	H
8	M	L	H	M	L	H	M	L	H	M	L	H

where:
"H" is the maximum (HI) signal, "M" is the mid-level signal, and "L" is the minimum (LO) signal.
See the QB Handbook for definitions of each signal for each assay format

Edge effects, which are not uncommon in this type of assay, will be seen by comparing min and max values throughout the 96 well plate. Consistent deviations in rows or columns will be noted, and should be corrected or accounted for before validation proceeds. The signal window size is also calculated and should be ≥2.

3. Response Surface Design: Example of Experimental Design to Develop and Optimize a C-ELISA

   To optimize the assay using experimental design it is best to work directly with a statistician. They will help in setting up the design and with analysis of the data.

• HUVEC cells with ERK p42/p44 Antibody
• Serum Starve time (0 - 4 hrs)
• VEGF conc. (originally 10-100. Changed to 30-200)
• Cell Density (2.5 - 10 x103 cells)
• Antibody conc. (1:2000, 1:1000, 1:500)

• Serum Starvation greater than 4 hours
• VegF conc. higher than 100 ng/ml
• Induction time less than 5 minutes
• Cell Density 20,00 cells per well

• Plating density: Cells at 10 or 20,000 cells per well
• Antibodies: Two antibodies pKDR (996) and pERK p42/44
• Culture conditions: Serum starvation for 4 hours or overnight

• HUVEC cells at 20,000 cells per well stimulated for 4 minutes at 150 ng/ml VEGF using 1:500 of pERK p42/44

After optimal conditions are determined test control compounds to determine if IC50’s can be obtained in the assay.

Follow this with plate to plate and within plate variability using the plate validation templates. If the assay passes plate validation then proceed with analyzing compounds in a test retest assay. See section XII of the QB manual for test retest.

Enzyme Inhibition: Western Analysis

Enzyme Inhibition: C-Elisa Analysis

Enzyme inhibitor profiling. A. Immunoblot analysis. Confluent HEK293 cells were serum-starved for 2h before incubating with increasing concentrations of specific enzyme inhibitors 1, 2 &3 for 1h. Where indicated, PMA was added for 10 min. Cell lysates were collected, electrophoresed and blotted. Blots were stained with a degenerate antibody to the specific enzyme phosphorylation consensus sequence that recognizes the phosphorylated form of several substrates in these cells. B. C-ELISA was performed on parallel cultures using duplicate wells per data point.

HEK293 cells were transfected with a specific enayme substrate using standard procedures. The next day, transfected and control (not transfected) cells were harvested and mixed together in the proportions indicated. The mixed populations were seeded into individual wells of a 96-well plate and cultured for an additional 24h. Cells were serum starved for 2 h in medium containing 0.1% BSA before adding a specific enzyme inhibitor. After additional 1 h incubation, PMA (200 nM) was added for 10 min. Cells were then processed according to the C-ELISA protocol. Phosphorylated substrate was measured using an antibody that selectively recognizes the exogenous enzyme substrate expressed only in the transfected cells.

1. http://www.licor.com/
2. http://www.activemotif.com/
3. http://www.selectscience.net/users-views/ttp-labtech/acumen-explorer
4. http://www.cellomics.com/