This method is useful for quantifying the early dynamics of cellular adhesion and spreading of anchorage-dependent cells onto the fibronectin. Furthermore, this assay can be used to investigate the effects of altered redox homeostasis on cell spreading and/or cell adhesion-related intracellular signaling pathways.
The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the homeostasis of adult tissues. Interestingly, oxidative stress can alter these processes, thus contributing to the pathophysiology of diseases such as metastatic cancer. Therefore, understanding the mechanism(s) of how cells attach and spread on the ECM during perturbations in redox status can provide insight into normal and disease states. Described below is a step-wise protocol that utilizes an immunofluorescence-based assay to specifically quantify cell adhesion and spreading of immortalized fibroblast cells on fibronectin (FN) in vitro. Briefly, anchorage-dependent cells are held in suspension and exposed to the ATM kinase inhibitor Ku55933 to induce oxidative stress. Cells are then plated on FN-coated surface and allowed to attach for predetermined periods of time. Cells that remain attached are fixed and labeled with fluorescence-based antibody markers of adhesion (e.g., paxillin) and spreading (e.g., F-actin). Data acquisition and analysis are performed using commonly available laboratory equipment, including an epifluorescence microscope and freely available Fiji software. This procedure is highly versatile and can be modified for a variety of cell lines, ECM proteins, or inhibitors in order to examine a broad range of biological questions.
Cell-matrix adhesions (i.e., focal adhesions) are large and dynamic multimolecular protein complexes which mediate cell adhesion and spreading. These processes are critical for tissue development, maintenance, and physiological function. Focal adhesions are composed of membrane-bound receptors, such as integrins, as well as scaffolding proteins that link cytoskeletal actin to the extracellular matrix (ECM)1. These complexes are capable of responding to physiochemical cues present in the extracellular environment through the activation of various signaling transduction pathways. As such, focal adhesions serve as signaling centers to propagate extracellular mechanical cues into a number of cellular processes including directed migration, cell cycle regulation, differentiation, and survival1,2. One group of signaling molecules that regulate and interact with focal adhesions includes members of the Rho family of small GTPases. Rho GTPases are key proteins that regulate cell migration and adhesion dynamics through their specific spatiotemporal activation3. Not surprisingly, dysregulation of Rho protein function has been implicated in a number of human pathologies such as metastasis, angiogenesis, and others. Of particular interest, cellular redox status plays a predominant role in the modulation of cell migration and adhesion. Alterations in redox homeostasis, such as increases in reactive oxygen species (ROS), have been demonstrated to regulate Rho protein activity, as well as adhesion, in a number of cell types and human diseases4,5,6,7,8. For example, individuals suffering from the neurological disorder ataxia-telangiectasia (A-T), which is caused by a mutation in the DNA damage repair serine/threonine kinase A-T-mutated (ATM), have an increased risk of metastatic cancer9,10. Loss of ATM kinase activity in these patients and cell lines, either through genetic mutation or chemical inhibition, results in high levels of oxidative stress due to dysfunction of the pentose phosphate pathway7,11,12. Moreover, recent studies from the laboratory have highlighted a pathophysiological role for ROS in A-T by altering cytoskeletal dynamics (i.e. adhesion and spreading) as a direct result of activating Rho family GTPases in vitro5. Ultimately, these alterations in cytoskeletal dynamics caused by Rho family activation may lead to the increased risk of metastatic cancer noted in A-T patients5,13. Therefore, understanding the interplay between cell-matrix interactions during oxidative stress can provide insights into the regulation of adhesion and spreading. These studies can also set the stage for further investigations into a possible role for Rho family GTPases in these signaling processes.
Described herein is a protocol to study the early cellular dynamics of adhesion assembly and spreading during oxidative stress caused by inhibition of ATM kinase activity. This assay is based on the well-characterized mechanism of anchorage-dependent cells adhesion to the ECM protein fibronectin (FN). When cells maintained in suspension are plated onto FN, several Rho GTPases coordinate the control of the actin cytoskeletal remodeling3,14. Morphological changes are observed as cells shift from round and circular in appearance to flattened and expanded. Concomitant with these observations is the development of numerous matrix adhesions with the ECM. These changes are attributed to the biphasic activation of RhoA with Rac1 during the first hour as cells adhere and spread 15,16.
A variety of methods have been utilized to examine adhesion morphology and dynamics as well as cell spreading. However, these methods rely on sophisticated long-term, live-imaging total internal reflection fluorescence (TIRF) or confocal microscopy systems. Thus, users must have access to specialized equipment and software. Furthermore, the set-up time required by these bio-imaging systems makes capturing early adhesion events challenging, especially when testing multiple inhibitors or treatment conditions concurrently.
The methods detailed, herein, provide a straightforward, economical, yet quantitative way to assess parameters that govern the adhesion assembly and spreading in vitro. The protocol is performed using commonly available laboratory equipment, such as an epifluorescence microscope and CCD camera. This assay involves applying anchorage-dependent cells to an FN-coated surface after a period of oxidative stress caused by chemical inhibition of ATM kinase activity, which has been demonstrated previously5. Following plating, cells are allowed to attach and adhere for specified lengths of time. Unattached cells are washed away, while attached cells are fixed and labeled with fluorescence-based antibodies to markers of adhesion (e.g., paxillin) and spreading (e.g., F-actin)2,5. These proteins are then visualized and recorded using an epifluorescence microscope. Subsequent data analysis is performed using freely available Fiji software. Moreover, this method can be adapted to examine adhesion dynamics under a wide range of conditions including different ECM proteins, treatment with various oxidants/cell culture conditions or a variety of anchorage-dependent cell lines to address a broad range of biological questions.
1. Preparations
NOTE: The protocol described below has been optimized for the use with REF52 cells and ATM+/+ or ATM-/- human fibroblasts. Other cell types may require further optimization as described in the notes and troubleshooting sections below.
2. Coating cell culture plates with the extracellular matrix protein fibronectin
NOTE: Perform this section using aseptic technique and sterile reagents in a BSL-2 certified laminar flow hood. Refer to Figure 1A for an overview of key steps prior to beginning.
3. Preparing anchorage-dependent cells for the adhesion assay
NOTE: Perform this section using aseptic technique and sterile reagents in a BSL-2 certified laminar flow hood.
4. Cell fixation and antibody staining for immunofluorescence
NOTE: The following steps are performed under non-sterile conditions and at room temperature unless otherwise stated.
5. Quantifying stress fibers, cell circularity, and focal adhesion formation
NOTE: The following image analyses are performed using the latest version of the open source imaging processing package Fiji Is Just Image J (Fiji), which can be downloaded free of charge at (http://fiji.sc/).
A general schema of the experimental set-up
Figure 1 represents the general schema for the cell adhesion and spreading protocol beginning with serum starvation of REF52 cells and ending with computational analysis of acquired fluorescence images. Key steps in the protocol are illustrated in the timeline. Of note, step 2 of the protocol describes the preparation of the FN-coated coverslips, which should be performed concurrently with step 3: serum starving REF52 cells prior to placing them in suspension (Figure 1A). An example of a mock-labeled 24-well plate indicating treatment groups and duration of cell adhesion prior to fixation of samples for fluorescence microscopy (Figure 1B).
Immunofluorescence image processing for focal adhesion quantification
REF52 cells were held in suspension for 90 minutes, plated on FN, and allowed to adhere for an additional 15 minutes. After fixation and staining with an anti-paxillin antibody, fluorescent 8-bit grayscale images of the cells were acquired. Image processing analysis was performed according to the protocol delineated in Step 5. Shown are representative images of each distinct processing step including the original image (Figure 2A), and images following background subtraction (post-Rolling Ball) (Figure 2B), CLAHE (Figure 2C), Gaussian Blur (Figure 2D) and Mexican Hat Filter (Post-MHF) (Figure 2E) filtering steps. After completing all image processing steps, individual focal adhesions should be prominent, in focus, and readily distinguishable from one another (Figure 2E). After the images are filtered, the focal adhesions can be quantified and their area measured (steps 5.1 and 5.4).
Visualization of cell adhesion and spreading on FN after oxidative stress
A representative grayscale fluorescence image of anti-paxillin (focal adhesion marker) (Figure 3, top panel) and phalloidin F-actin probe staining (Figure 3, bottom panel) of REF52 cells after plating on FN with or without Ku55933 (ROS-inducing agent) treatment. Prior to the assay, REF52 cells were serum starved for 1 h. Following serum starvation, cells were held in suspension while being treated with either vehicle alone or 10 μM Ku55933 to induce oxidative stress. Cells were plated on FN-coated coverslips for the indicated times, fixed, and then stained with an antibody to focal adhesions and phalloidin to detect F-actin proteins. Prominent focal adhesions and stress fibers should be readily visible in REF52 cells after being allowed to adhere for 20-30 min on FN. Plated cells should not overlap with one another to permit full cellular spreading after adhesion. Notice the clear, distinct cell edges as well as space for individual cells to spread (Figure 3). F-actin enriched ruffles at the leading edge of cell membranes are visible and indicated with an arrow (Figure 3, bottom panel).
Graphical representation of quantified fluorescence images of stress fibers and the degree of cell spreading
Examples of quantified images displayed in bar graph form representing the percentage of cells with stress fibers and the degree of cell spreading with and without Ku55933 treatment at various times after adhesion. Fluorescent images of phalloidin F-actin probe and anti-paxillin staining, similar to images shown in Figure 3, were analyzed for the percentage of stress fibers and cell spreading (i.e., circularity index) using the image analysis procedures described in step 5 of the protocol. Notably, oxidant treatment caused a significant increase in stress fiber formation at all adhesion time points examined (Figure 4A) and a decrease in cell spreading following 15 minutes of cell adhesion to FN (Figure 4B).
Non-quantifiable immunofluorescence images due to cellular over confluency
Serum-starved REF52 cells were held in suspension for 90 minutes, during which time they were treated with 10 μM Ku55933 to induce ROS formation. Cells were then plated on FN and allowed to adhere for 20 minutes, after which they were fixed and stained with anti-paxillin or phalloidin-Alexa 594 F-actin probe. Plating at higher cell densities leads to cellular crowding, which prohibits cells from fully spreading due to over confluency. Notice cell edges are indistinguishable from adjacent cells (yellow arrows) (Figure 5A). As a result, quantification of individual cells is precluded, and spreading circumference cannot be accurately determined. In Figure 5B, a separate cell line, mouse embryonic fibroblasts (MEF), were held in suspension and then plated on FN for 30 minutes. Cells were then fixed and stained with an anti-paxillin antibody. Out of focus cells are denoted by red arrows (Figure 5B). Furthermore, the cross-reactivity of the anti-paxillin antibody with cellular debris (blue arrow) will alter thresholding during quantitative image analysis (Part 5) and should not be included in the analysis (Figure 5B).
Figure 1: Time-line of protocol and example 24-well plate set-up.
(A) The time-line highlights key steps in the cell adhesion and spreading procedure. (B) Representative labeled 24-plate, illustrating treatment groups and times for cell adhesion. Please click here to view a larger version of this figure.
Figure 2: Examples of representative immunofluorescence images following image-processing.
REF52 cells were held in suspension for 90 min, plated on FN, and allowed to adhere for 15 min. Cells were fixed and stained with an anti-paxillin antibody. (A) Original image and images following (B) Background subtraction (post-Rolling Ball), (C) CLAHE, (D) Gaussian Blur and (E) Mexican Hat Filter (Post-MHF) filtering steps. Bar, 20 μm. Please click here to view a larger version of this figure.
Figure 3: Representative immunofluorescence images of anti-paxillin and phalloidin F-actin probe stained REF52 cells plated on FN.
Prior to the assay, REF52 cells were serum starved for 1 h. Following serum starvation, cells were held in suspension while treated with either vehicle alone or 10 μM Ku55933 to cause oxidative stress. Cells were plated on FN-coated coverslips for the indicated times, fixed and stained with an antibody to focal adhesions and phalloidin to detect F-actin proteins. F-actin enriched ruffles at the leading edge of cell membranes are indicated with an arrow. Bar, 40 μm. This figure has been modified from Tolbert et al.5 Please click here to view a larger version of this figure.
Figure 4: Quantification of immunofluorescence images.
Graphs illustrating (A) the percentage of cells exhibiting stress fibers and (B) cell circularity measurements. Cell circularity was defined as the cell area divided by the cell perimeter. Values ranged from 0-1.0 indicating an elongated or rounded morphology, respectively. Error bars indicate S.E.M. Student’s t-test for paired samples *p<0.01 from experiments performed in triplicate. This figure has been modified from Tolbert et al.5 Please click here to view a larger version of this figure.
Figure 5: Non-quantifiable immunofluorescence images.
(A) Serum-starved REF52 cells were held in suspension for 90 min, while treated with 10 μM Ku55933. Cells were plated on FN and allowed to adhere for 20 min. Cells were fixed and stained with anti-paxillin or phalloidin-Alexa 594 F-actin probe. Cell edges are shown by yellow arrows. (B) MEF cells were held in suspension and then plated on FN for 30 min. Cells were fixed and stained with an anti-paxillin antibody. Out of focus cells are denoted by red arrows and cross-reactivity of anti-paxillin antibody with cellular debris are denoted with blue arrows. Bar, 30 μm. Please click here to view a larger version of this figure.
The protocol described here is a versatile and economical way to rapidly screen a number of anchorage-dependent cell types for dynamic cytoskeleton remodeling during cell spreading. In particular, this method quantitatively examines stress fiber and focal adhesion formation during oxidative stress when cells adhere to FN (Figure 1A). Moreover, these cellular phenotypes may suggest a regulatory role for members of the Rho family of small GTPases since they have documented roles during cell attachment and spreading15,16,22. However, additional biochemical techniques would be required to identify the possible involvement of GTP-bound, active Rho family proteins.
The presented protocol utilizes immunofluorescence detection of F-actin and paxillin to specifically examine cell adhesion and spreading of immortalized fibroblast cells on FN after oxidative stress induced by ATM kinase inhibition (Figure 2, Figure 3 and Figure 4). However, this procedure can also be adapted for use on other ECM proteins and/or for other adherent cell types. When adapting to other cell lines, it is important to optimize the experimental conditions, particularly: cell number/density, time of serum starvation, ECM protein concentration, and oxidative stress treatment conditions. When testing the effects of an unknown stimulus on adhesion and spreading dynamics, it is necessary to include both negative and positive control samples to verify that the assay is functioning correctly. Negative control samples can include an untreated or vehicle-only sample, while a positive control should induce oxidative stress (e.g., H2O2). Furthermore, although not discussed here, it is also essential to utilize the proper antibody controls. It is recommended that three separate controls be used for each antibody to verify its specificity and to identify any potential fluorescence bleed-through23,24. These include: 1) a primary antibody control to ensure specific binding of the primary antibody to the antigen and to confirm that antigen binding occurs under the fixation conditions used, 2) a secondary antibody control (for non-secondary conjugated-antibodies) that shows specificity to the primary antibody, and 3) fluorophore controls that ensure the fluorophore added is not the result of endogenous fluorescence or bleed-through from another antibody.
While this assay is useful for analyzing the early kinetic events of adhesion assembly and spreading, it is not suitable for the detailed examination of adhesion disassembly or adhesion strength and cellular reinforcement. The latter requires the use of long-term imaging bio-stations or single-cell force spectroscopy techniques. The latter techniques include atomic-force microscopy, optical tweezers, tension sensors of adhesion proteins such as vinculin25 or talin26,27, and 3D-force microscopy28.
Critical steps in the protocol include thoroughly coating coverslips with FN. This is necessary for the uniform spreading and adhesion of cells. It is therefore important to pipette the FN solution up and down over the coverslips multiple times prior to incubation. Coverslips must remain fully submerged in the FN solution during the incubation time. FN coated coverslips can be stored for up to 2 weeks at 4 °C.
Cell density is also important, as cells that are plated too densely will not achieve maximum spreading circumference. Furthermore, it would not be possible to distinguish individual focal adhesions or cellular ruffles for each individual cell. It is, therefore, necessary to count the cells using a hemocytometer or automated cell counter prior to placing the cells in suspension. While an estimate of cell density is provided for REF52 cells, this will need to be empirically determined for other cells under study. Cells should be plated sparsely enough that few cells overlap, allowing them to spread fully (Figure 5).
Other critical steps in the protocol to consider are fluorescently conjugated phalloidin-Alexa 594 F-actin probe and secondary antibodies are light sensitive. Therefore, samples should be minimally exposed to light after the application of these reagents. Furthermore, a number of agents that induce oxidative stress have short half-lives. It is, therefore, necessary to test the chosen oxidant for optimal dose and exposure time to achieve peak activity.
The following sections contain trouble-shooting tips with regards to FN concentration, serum starvation conditions, and cell detachment methodologies. These tips are useful when adapting the protocol for other cells lines and/or treatment conditions.
For consistent FA analysis, optimal attachment and spreading conditions are necessary, which will vary depending upon the ECM ligand. For FN, begin using a dynamic range of 10-30 μg/mL. At higher concentrations of FN, there is little difference in cell attachment. However, some cell types do not efficiently spread at higher concentrations of ECM.
Each cell type responds differently to conditions of serum starvation. REF52 cells can easily be starved overnight without any loss of viability, however, this is not true for all cell lines. Therefore, it is necessary to determine the degree to which the cell line under study can withstand serum starvation.
For spreading assays, cells need to be trypsinized for as little time as possible for reproducible results29. Following cell detachment by trypsinization, cell surface receptors, their cognate ligands, and Rho GTPase activity require a recovery period to return to steady-state levels14,15. The trypsinization procedure outlined in this protocol should enable cells to retain most of their cell surface FN-binding receptors29. However, certain cell types may require alternative methods of cell detachment to completely prevent digestion of cell surface receptors. These methods include chelating cells using EDTA-based solutions (e.g., Versene) or milder enzymatic dissociation solutions (e.g., Accutase). Use of these dissociation solutions may leave cell-cell adhesions intact resulting in cell clumps. It is therefore important to completely resuspend individual cells to ensure experimental reproducibility.
The authors have nothing to disclose.
The authors thank Drs. Scott R. Hutton and Meghan S. Blackledge for the critical review of the manuscript. This work was funded by High Point University’s Research and Sponsored Programs (MCS) and the Biotechnology Program at North Carolina State University (MCS).
0.05% Trypsin-EDTA (1x) | Gibco by Life Technologies | 25300-054 | cell dissociation |
10 cm2 dishes | Cell Treat | 229620 | sterile, tissue culture treated |
15 mL conical tubes | Fisher Scientific | 05-539-5 | sterile |
1X Phosphate Buffered Saline | Corning Cellgro | 21-031-CV | PBS, sterile, free of Mg2+ and Ca2+ |
24-well cell culture treated plates | Fisher Scientific | 07-200-740 | sterile, tissue culture treated |
4°C refrigerator | Fisher Scientific | ||
Mouse IgG anti-paxillin primary antibody (clone 165) | BD Transduction Laboratories | 610620 | marker of focal adhesions |
Aspirator | Argos | EV310 | |
Biosafety cabinet | Nuair | NU-477-400 | Class II, Type A, series 5 |
Delipidated Bovine Serum Albumin (Fatty Acid Free) Powder | Fisher Scientific | BP9704-100 | dlBSA |
Dimethyl Sulfoxide | Fisher Scientific | BP231-100 | organic solvent to dissolve Ku55933 |
Dulbecco's Modified Eagle Media, High Glucose | Fisher Scientific | 11965092 | REF52 base cell culture medium |
Fetal bovine serum | Fisher Scientific | 16000044 | certified, cell culture medium supplement |
Fiji | National Institutes of Health | http://fiji.sc/ | image analysis program |
Filter syringe | Fisher Scientific | 6900-2502 | 0.2 µM, sterile |
Glass coverslips (12-Cir-1.5) | Fisher Scientific | 12-545-81 | autoclave in foil to sterilize |
Goat anti-mouse IgG secondary antibody Alexa Fluor 488 | Invitrogen | A11001 | fluorescent secondary antibody, light sensitive |
Goat Serum | Gibco by Life Technologies | 16210-064 | component of blocking solution for immunofluorescence |
Hemocytometer | Fisher Scientific | 22-600-107 | for cell counting |
Human Plasma Fibronectin | Gibco by Life Technologies | 33016-015 | FN |
IX73 Fluorescence Inverted Microscope | Olympus | microscope to visualize fluorescence, cell morphology, counting and dissociation | |
Ku55933 | Sigma-Aldrich | SML1109-25MG | ATM kinase inhibitor, inducer of reactive oxygen species |
L-glutamine | Fisher Scientific | 25-030-081 | cell culture medium supplement |
Monochrome CMOS 16 bit camera | Optimos | ||
Paraformaldehyde | Sigma-Aldrich | P6148-500G | PFA, fixative for immunofluorescence |
Penicillin-streptomycin | Fisher Scientific | 15-140-122 | P/S, antibiotic solution for culture medium |
Alexa Fluor 594 phalloidin (F-actin probe) | Invitrogen | A12381 | marker of F-actin, light sensitive |
ProLong Gold Anti-fade reagent with DAPI | Invitrogen | P36941 | cover slip mounting media including nuclear dye DAPI, light sensitive |
REF52 cells | Graham, D.M. et. al. Journal of Cell Biology 2018 | ||
Stir plate with heat control | Corning Incorporated | PC-420D | |
Syringe | BD Biosciences | 309653 | 60 mL syringe |
Tissue culture incubator | Nuair | ||
Triton X-100 | Fisher Scientific | BP151-500 | detergent used to permeabilize cell membranes |
Trypan Blue Solution | Fisher Scientific | 15-250-061 | for cell counting |
Trypsin Neutralizing Solution (1x) | Gibco by Life Technologies | R-002-100 | TNS, neutralizes trypsin instead of fetal bovine serum |
tube rotator | Fisher Scientific | 11-676-341 | |
water bath | Fisher Scientific | FSGPD02 |