On-chip, cell-based microarray immunofluorescence assay for high-throughput analysis of target proteins.

Cell Culture

Upon thawing, Mia PaCa-2 human pancreatic tumor cells (ATCC, Manassas, VA) were grown in T75 tissue culture flasks until confluence at 37 °C in a 5% CO 2 incubator (HEPA class100, Thermo, Waltham, MA), using low-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 1% (v/v) penicillin–streptomycin, and 1% (v/v) MEM nonessential aminoacids, all from Invitrogen (Carlsbad, CA). The cells were either trypsinized and seeded in six-well plates (1 × 10 5 cells/mL) or used for cell spotting on functionalized glass slides. After each passage, viable and dead cells were determined by counting in a hemocytometer using an optical microscope and the trypan blue dye (Invitrogen) exclusion test.

Western Blotting Analysis of HIF-1α

Levels in Mia PaCa-2 Cells Mia PaCa-2 cells were used to assess the expression of HIF-1α. The cells were seeded in six-well plates (1 × 10 5 cells/mL) and grown until confluence at 37 °C in a 5% CO 2 incubator. The medium was replaced every 48 h. The hypoxia-mimicking agent carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132, EMD Chemicals, Gibbstown, NJ) was used for induction of HIF-1α expression and 2-methoxyestradiol (2ME2, Sigma, St. Louis, MO) was used for inhibition of HIF-1α. A whole-cell extract was prepared by lysing the cells with 100 mM potassium phosphate (pH 7.8) and 0.2% (v/v) Triton X-100 supplemented with protease and phosphatase inhibitors. After cell lysis, the lysate was collected and equal amounts of protein were loaded in each lane. The samples were resolved in a 7.5% precast SDS-PAGE gel for 1 h at 100 V and electrotransferred to a cellulose membrane, both from Bio-Rad (Hercules, CA). The membrane was then immersed in blocking buffer (5% (w/v) skim milk and 0.05% (v/v) Tween-20 in phosphate-buffered saline (PBS)) overnight with agitation. The primary antibody (mouse monoclonal anti-HIF-1α, BD Biosciences, San Jose, CA) was diluted (1:250, v/v) in the blocking buffer and incubated with the membrane for 1 h at room temperature. The membrane was then washed three times for 10 min with 0.05% (v/v) Tween-20 in PBS (PBS-T) and incubated with the secondary antibody (peroxidase-conjugated goat anti-mouse IgG, Pierce, Rockford, IL) (1:1000 dilution in blocking buffer) overnight at 4 °C. The membrane was again washed three times for 10 min with PBS-T and immunoreactivity was visualized using a chemiluminescence reagent (SuperSignal, Pierce). β -Actin was used as internal control (rabbit monoclonal anti- β -actin, Cell Signaling Technology, Danvers, MA). Glass Slide Modification Borosilicate glass slides (25 × 75 mm 2 , Fisher, Pittsburgh, PA) were prewashed with ethanol followed by acid treatment in concentrated sulfuric acid (98%) overnight to remove dust and oil from the glass surface. The slides were then sonicated for 30 min and rinsed in deionized distilled water five times and then once in acetone. The cleaned glass slides were dried using a nitrogen stream and then baked at 120 °C for 15 min prior to use. To generate slides for cell spotting the acid-cleaned glass slides were spin-coated (WS-400B-6NPP/Lite, Laurell Co., North Wales, PA) with 0.1% (w/v) poly (styrene- co -maleic anhydride) (PS-MA, Sigma) in toluene solution as described previously. 4 Briefly, the 0.1% (w/v) PS-MA solution was prepared starting from a 1% (w/v) stock solution, and 1.5 mL was spin-coated on top of a clean glass slide surface at 3000 rpm for 30 s. Methyltrimethoxysilane (MTMOS, Sigma)-coated slides were also prepared as described previously. 3 These slides were used for stamping MG-132 and 2ME2 in the cell spots to perform HIF-1α induction/inhibition studies on the microarrays. Cell Spotting A poly-L-lysine (PLL)-Ba 2+ mixture was prepared by mixing a solution of BaCl 2 in water (0.1 M) and sterile PLL (0.01% (w/v)), both from Sigma, in a 1:2 volume ratio, respectively. This mixture was spotted on the PS-MA-coated glass slides using a MicroSys 5100–4SQ noncontact microarray spotter (Genomic Solutions, Ann Arbor, MI). A mixture of low-viscosity alginate (Sigma) and cell suspension in DMEM (supplemented with 10% FBS) was prepared so that the final concentrations of cells and of alginate were 6 × 10 6 cells/mL and 1% (w/v), respectively. During cell spotting, the humidity within the microarrayer chamber was maintained above 90% to retard water evaporation from the applied spots. To minimize potential microbial contamination, the microarray spotter chamber was sterilized with 70% ethanol prior to use, and growth medium was supplemented with antibiotics (penicillin and streptomycin).

Cellular Viability Assay

The spotted slides were immersed in DMEM/10%FBS complete medium and incubated at 37 °C in a 5% CO 2 incubator for 5 days. Medium was changed after 48 and 96 h. Cellular viability was assessed every day with the LIVE/DEAD Viability/Cytotoxicity Kit for mammalian cells (Invitrogen). Briefly, the slides were rinsed three times in PBS and a solution containing 0.5 μ M calcein AM was used to detect viable cells through green fluorescence intensity. The fluorescence was scanned and quantified from the scanning image using GenePix Pro 6.0 (Molecular Devices Co., Sunnyvale, CA). The fold increase in total viable cell number was calculated by the ratio of the fluorescence intensity at each day and the fluorescence intensity at day 0. Mimicking Hypoxia in Cellular Microarray Spotted slides were immersed in 10% FBS-supplemented DMEM medium and incubated for 3–4 days at 37 °C in a 5% CO 2 incubator. Several dilutions of the hypoxia mimicking agent MG-132 were made by adding this agent to the culture medium, followed by 6-h incubation. The slides were then processed, and the levels of HIF-1α were detected using the in situ immunofluorescence assay. Slides incubated under normoxic conditions (no MG-132 added) were used for comparison. To show the potential of this platform for screening small molecules and their effects in cell models, we dispensed 60 nL of known concentrations of MG-132 and 2ME2 onto MTMOS-coated slides. The resulting array was immediately stamped on top of the 3D cellular array, such that each compound-containing spot contacted a cell spot. 4 A silicone gasket was used to maintain proper spacing between the two slides. The dual slide system was subsequently incubated for 6 h at 37 °C in a nearly 100% humidified hybridization chamber (ArrayIt, Sunnyvale, CA) to prevent evaporation. After stamping, the cell spots were rinsed and the cellular microarray was analyzed using the in-cell, chip-based Western assay.

Cellular Microarray-Based Immunofluorescence Assay

Following incubation the slides were rinsed three times in washing buffer (PBS containing 20 mM CaCl 2 ) for at least 15 min, followed by fixation with a solution of methanol and acetone (1:1, v/v) for 20 min at −20 °C. The slides were then rinsed twice in washing buffer and incubated overnight in blocking solution (SuperBlock, Pierce). The primary antibody (mouse anti-HIF-1α, 1:500 dilution in PBS-T containing 1% (w/v) bovine serum albumin (BSA)) was then added to the slides, and the cells were incubated overnight at 4 °C. The slides were then washed three times in washing buffer for 15 min each, the secondary antibody (peroxidase-conjugated goat-anti mouse IgG, Molecular Probes (Carlsbad, CA), 1:1000 dilution in PBS-T containing 1% (w/v) BSA) was added to the slides, and the cells were incubated for 3 h at room temperature. A tyramide signal amplification kit (Molecular Probes) was used following the manufacturer’s instructions to detect the presence of the protein HIF-1α through fluorescence analysis. The fluorescence was measured with the slide scanner and the images of stained cells were analyzed using the program GenePix Pro 6.0. The fluorescence signal of β -actin (determined by the same protocol using triplicate slides for each condition) was used as the internal control.

Western Blotting Analysis of HIF-1α

Levels in Mia PaCa-2 Cells Mia PaCa-2 cells were used to assess the expression of HIF-1α. The cells were seeded in six-well plates (1 × 10 5 cells/mL) and grown until confluence at 37 °C in a 5% CO 2 incubator. The medium was replaced every 48 h. The hypoxia-mimicking agent carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132, EMD Chemicals, Gibbstown, NJ) was used for induction of HIF-1α expression and 2-methoxyestradiol (2ME2, Sigma, St. Louis, MO) was used for inhibition of HIF-1α. A whole-cell extract was prepared by lysing the cells with 100 mM potassium phosphate (pH 7.8) and 0.2% (v/v) Triton X-100 supplemented with protease and phosphatase inhibitors. After cell lysis, the lysate was collected and equal amounts of protein were loaded in each lane. The samples were resolved in a 7.5% precast SDS-PAGE gel for 1 h at 100 V and electrotransferred to a cellulose membrane, both from Bio-Rad (Hercules, CA). The membrane was then immersed in blocking buffer (5% (w/v) skim milk and 0.05% (v/v) Tween-20 in phosphate-buffered saline (PBS)) overnight with agitation. The primary antibody (mouse monoclonal anti-HIF-1α, BD Biosciences, San Jose, CA) was diluted (1:250, v/v) in the blocking buffer and incubated with the membrane for 1 h at room temperature. The membrane was then washed three times for 10 min with 0.05% (v/v) Tween-20 in PBS (PBS-T) and incubated with the secondary antibody (peroxidase-conjugated goat anti-mouse IgG, Pierce, Rockford, IL) (1:1000 dilution in blocking buffer) overnight at 4 °C. The membrane was again washed three times for 10 min with PBS-T and immunoreactivity was visualized using a chemiluminescence reagent (SuperSignal, Pierce). β -Actin was used as internal control (rabbit monoclonal anti- β -actin, Cell Signaling Technology, Danvers, MA).