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High-Throughput Imaging and Automated Analysis of Focal AdhesionsDownload
Related Products: Cytation C10
August 01, 2022
Using the Agilent BioTek Cytation C10 confocal imaging reader
Authors: Ernest Heimsath, PhD and Peter J. Brescia, MSc. Agilent Technologies, Inc. Winooski, VT, USA
Focal adhesions (FAs) are intra and extracellular multiprotein complexes that act as attachment points between the plasma membrane of a cell and its surrounding extracellular matrix (ECM). FAs localize to a narrow z range on the ventral side of cells. Therefore, imaging them is challenging without specialized microscopy techniques and equipment that is not conducive with high-throughput imaging. This application note demonstrates that the Agilent BioTek Cytation C10 confocal imaging reader, combined with the Agilent BioTek Gen5 microplate reader and imager software, can reliably image and identify FAs in a high-throughput, 96-well format. This expanded throughput provides new possibilities to screen cancer drugs targeted for FAs.
Introduction Cell migration is crucial for a multitude of physiologic processes and requires that cells interact with their surrounding environment.1 FAs are multiprotein complexes that act as attachment points between the plasma membrane of a cell and the surrounding ECM, and are crucial for cell migration.1, 2 FAs are central for normal biological processes involving cell migration, such as embryogenesis, tissue regeneration, and the inflammatory cell response to sites of infection.1 FAs are also integral for cancer metastasis.3, 4 Because FAs localize to a very narrow ventral z range of the cell, they are typically imaged using total internal reflection fluorescence (TIRF) microscopy, which eliminates background signal contributed from the rest of the cell volume. While this imaging technique is powerful, it is often not conducive to applications designed for high-throughput formats, such as drug titrations in a 96-well microplate. This application note demonstrates that the Cytation C10 confocal imaging reader, combined with Gen5 software, can reliably image and identify focal adhesions in a 96-well format. An expanded throughput provides new possibilities to screen cancer drugs targeted for focal adhesions.
Immortalized human fibroblasts (CI-huFIB, part number INSCI-1010) were a gift from InSCREENeX GmbH (Braunschweig, Germany) and were cultured in huFIB medium (part number INS-ME-1001). COS-7 cells were purchased from ATCC (part number CRL-1651; Manassas, VA) and maintained in advanced DMEM containing 10% FBS and 1x penn/strep/ glutamine.
Glass-bottom (170 µm thickness) 96-well microplates (part number 655891; Greiner Bio-One, Monroe, NC) were coated with 10 ng/µL fibronectin (part number F1141; SigmaAldrich, St. Louis, MO) as follows: wells were treated with 2N HCl (diluted with dH2 O from 12N stock) for 30 minutes, followed by three washes with sterile dH2 O. Poly-L-lysine (Sigma-Aldrich), 50 µg/mL, was then added to each well and incubated for 20 minutes at room temperature (RT). After three washes with sterile DPBS (Gibco), wells were treated with 0.5% glutaraldehyde for 15 minutes. Following another three washes with sterile DPBS, wells were treated for 15 minutes with fibronectin diluted to 10 ng/µL in DPBS.
After three more washes with DPBS, residual glutaraldehyde was deactivated by washing wells three times with DPBS containing 0.3 M glycine for at least 15 minutes. Wells were then washed three times with DPBS before cells were seeded.
Cell seeding and immunostaining procedure
Cells were seeded in 96-well plates at a density of 3.5 × 103 cells/well (huFIB) or 2 × 103 cells/well (COS-7). The cells were allowed to settle and attach to the fibronectin-coated well bottom at RT for one hour. Plates were transferred to a TC incubator and incubated overnight at 37 °C. Cells were fixed with 4% paraformaldehyde for 15 minutes, followed by three washes with PBS containing 0.3 M glycine. Cells were then permeabilized for five minutes with 0.5% Triton X-100, followed by blocking with 5% BSA for 30 minutes. Cells were incubated for 30 minutes with primary antibodies against paxillin (0.25 µg/mL; part number ab32084; Abcam, Cambridge, UK) and β-tubulin (1 µg/mL; EnCor Biotechnology Inc.; part number MCA-1B12) diluted in 5% BSA containing 0.1% Tween 20. After primary antibody incubation, wells were washed three times with PBS containing 0.1% Tween 20. Wells were incubated with Hoechst 34580 (250 nM; Thermo Fisher Scientific, Waltham, MA), CF633-conjugated phalloidin (1:40 dilution; Biotium, Fremont, CA) and secondary antibodies prespun at 14 K rpm for 10 minutes. For paxillin, Alexa 555-conjugated antirabbit IgG F(ab’)2 was used (Cell Signaling Technology, Danvers, MA), and Alexa 488-conjugated antimouse F(ab’)2 IgG for β-tubulin. After three washes with PBS + 0.1% Tween 20 and two washes with PBS, cells were immersed in imaging media (80% glycerol, 20 mM tris pH 8.0, 0.5% n-propyl gallate, and 0.05% sodium azide).
Imaging focal adhesion in a 96-well plate format
Focal adhesions were imaged using a 60x 0.7 NA air objective (Olympus Corporation, Tokyo, Japan). Due to the narrow z range where focal adhesions are found, during automated imaging it was crucial to optimize the z-stack range accordingly. Laser-based autofocus produced the most consistent results when capturing the optimized z range. The z-stack range was set to 3 µm, with one slice below the set focal point. For processing, z projections of the focal adhesion channel were limited to a 1.8 µm z range. However, the channel used for the primary mask included the entire z range, so that maximum signal could be achieved to create a more reliable primary mask.
Results and discussion
Confocal mode provides improved contrast to focal adhesions.
FAs are transient protein complexes located within a narrow z range (80 nm) at the ventral side of a cell.2 Furthermore, FA-associated components are not limited to FAs, but can localize elsewhere within the cytoplasm. When imaging FAs by fluorescently labeling any of these components, the ensuing nonFA-specific signal is exacerbated with widefield microscopy (Figure 1D). Using the Cytation C10 confocal imaging reader in confocal mode, FAs of huFIBs were successfully imaged when immunoprobed for paxillin, a known component of FAs. Additionally, cells were counterstained with phalloidin (F-actin) and Hoechst 34580 (nucleus) (Figure 1A and B). The huFIB cells were selected as they are an immortalized primary cell line carefully engineered to recapitulate the nonpathologic cellular phenotype of fibroblasts.5 Z-stacks of cells were taken at 60x with step sizes that met Nyquist sampling. Maximum intensity z projections were generated from a 1.8 µm range where FA signal was the most intense. This approach dramatically reduced background signal while improving FA resolution (Figure 1C, E, and G) compared to widefield (Figure 1D, F, and H).
Figure 1. The confocal mode of the Agilent BioTek Cytation C10 confocal imaging reader provides improved resolution of FAs. The huFIB cells were stained with antipaxillin (FAs; red), phalloidin (F-actin; greyscale), and Hoechst 34580 (nucleus; blue). The top row of images (A, C, E, and G) shows a cell captured in confocal mode. The bottom row (B, D, F, and H) shows images of the same cell captured in widefield mode. (A and B) Z projection of the ventral side of huFIB cells displaying FAs, F-actin, and nucleus. (C and D) The FA channel alone is shown. To better visualize signal, the raw TIF image was converted to the Fire LUT in ImageJ, which correlates signal intensity with the color spectrum; blue indicates low signal and red indicates high signal. (E to H) Close-up view of the two regions indicated with the boxes, which highlights the superior resolution of FAs using confocal mode. Images E and F are close ups of the yellow box in image C, whereas images G and H are close ups of the yellow box in image D. Scale bars: (A to D) = 20 µm; (E to H) = 1 µm.
Quantifying focal adhesions using the Spot Counting module
Using Gen5, an automated method was developed to reliably identify and quantify FAs. COS-7 cells plated on fibronectin were fixed and immunoprobed with a paxillin antibody (red) and counterstained with phalloidin (F-actin; greyscale) and Hoechst 34580 (nucleus; blue) (Figure 2A). This property can be used to decipher between real FA signal and nonspecific signal. First, the cell boundary is defined by applying a primary mask using the phalloidin channel (Figure 2B). Using phalloidin as a counterstain is crucial in this context because FAs are linked to the ends of actin stress fibers. Their localization in relation to actin stress fibers can be used to validate the accuracy of FA identification using the Spot Counting module (Figure 2C). Once FAs are identified, they can be quantified as FAs per cell. However, in the case where cell density is greater than ideal, FAs per total cell area can also be used.
Figure 2. FAs can be identified and quantified using the Spot Counting module in Agilent BioTek Gen5 microplate reader and imager software. (A) Maximum z projection of two COS-7 cells immunoprobed for FAs (red) and counterstained with phalloidin (greyscale) and Hoechst 34580 (blue). (B) The phalloidin channel is used to create a primary mask (orange) around each cell. (C) Enlarged view of the box in (B) demonstrating that spots identified in the secondary mask localize to the ends of actin stress fibers, validating them as FAs. Scale bars: A to B = 20 µm; C = 5 µm.
The Cytation C10 enables high-throughput imaging and analysis of focal adhesions
The ability to reliably capture and quantify FAs by the Cytation C10 using a 60x air objective (0.7 NA) enables high-throughput screening. This is difficult to achieve with imaging methods otherwise employed to image FAs, such as TIRF microscopy using oil-immersion objectives. Although cytochalasin D does not directly target FA disassembly, it is a small molecule that effectively disrupts actin-based structures and, in effect, leads to FA destabilization. With cytochalasin D, a dilution series drug treatment was performed in a 96-well format using FAs/cell area as the readout. A four-parameter dose–response curve was successfully fit and an IC50 was derived for cytochalasin D on FA stability of 43.2 nM (Figure 3).
Figure 3. High-throughput dose–response curve of cytochalasin D on FA count. The IC50 of cytochalasin D on FA stability in huFIB cells was established in a 96-well format. FAs were scored as number of FA per cell area and are reported as a percentage of vehicle control (mean ± SD).
Due to their narrow z localization and high-signal background, focal adhesions are typically imaged with specialized microscopy techniques and equipment that is not conducive with high-throughput imaging. When imaging at or above the Nyquist sampling rate (z-step sizes), the Agilent BioTek Cytation C10 confocal imaging reader combined with the Agilent BioTek Gen5 microplate reader and imager software can reliably resolve and identify FAs in a 96-well format. An expanded throughput provides new possibilities to screen cancer drugs targeted for FAs.
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