Matrix Modifikasyon Bağlı Hücresel Yapışma ve De-yapışma Real-time Değişiklikleri Tedbir Hücre-substrat Empedans ve Canlı Hücre Görüntüleme kullanma

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

Hücre-matris yapışma hücre morfolojisini kontrol ve sinyalizasyon önemli bir rol oynar. Hücre-matris yapışma (örneğin, miyeloperoksidaz ve diğer matris düzenleyici oksidanlar / iltihap sırasında ortaya çıkan enzimler) bozan etmenler de hastalıkların bir dizi hücresel fonksiyonu, fenotip ve canlılığı patolojik değişiklikleri tetikleyerek söz konusu olmaktadır. Burada, (a patofizyolojik matris düzenleyici uyarıcı olarak endotel hücreleri ve myeloperoksidaz ile), hücre-alt-tabaka empedans ve canlı hücre görüntüleme teknikleri kolayca doğru hücre yapışmasının ve matris modifikasyonu ile indüklenen de yapışma gerçek zamanlı değişiklikleri ölçmek için kullanılabilir açıklanmıştır Yüksek zamansal çözünürlüğe sahip ve non-invaziv bir şekilde. XCELLigence hücre alt-tabaka empedans sistemi sürekli altın mikroelektrot diziler üzerinde yetiştirilen hücreler, hücre-alt-tabaka ara yüzeyinde elektrik empedansı ölçmek suretiyle hücre-matris yapışma alanını ifade etmektedir. Resim analizi zaman atlamalı ayırıcısıcaklık üzerindeki girişim zıtlık filmler hücre-matris temas alanında değişiklikleri temsil eden zaman içinde, tek tek hücrelerin öngörülen alan değişiklikleri ifade etmektedir. Her iki teknik doğru hücresel yapışma ve de-yapışma süreçlerine hızlı değişiklikler ölçmek. Mikroelektrot biyosensör dizileri Hücre-substrat empedans sağlam, yüksek hacimli ölçümler için bir platform sağlar. Canlı hücre görüntüleme hücre-substrat empedans ölçümleri ile sayısal morfolojik değişikliklerin doğası ve dinamikleri ile ilgili ek detay sağlamak analizleri. Bu tamamlayıcı yaklaşımlar hücre içi hücre dışı matris bileşenleri miyeloperoksidaz-katalize oksidatif modifikasyonu hızlı hücre yapışması, morfolojisi değişiklikler ve endotel hücreleri sinyal tetikler nasıl değerli yeni anlayışlar sağlar. Bu yaklaşımlar, diğer matris-modifiye uyarılara ve ilgili diğer yapışıcı hücreler (örneğin, epitelyal hücreleri) yanıt olarak hücre yapışma dinamiğini çalışmak için de geçerlidir.

Hücreler ve kendi çevre hücre dışı matris arasındaki kararlı yapışkan temas doku homeostazının muhafaza için gereklidir. Örneğin, kan damarlarında endotel altı matris endotelyal hücre yapışma endotelyal tabakanın bütünlüğünü ve düzenleme, yarı geçirgen bir bariyer vasküler ¹ olarak homeostatik fonksiyonunu korumada bir rol oynar. aktin hücre iskeleti mekanik merkezi yönlendirilmiş aktomizin çekme güçlerine karşı hücre zarı konumunu belirlemede önemli bir rol oynadığı hücre-matris arayüz de hücre-matris yapışma ve yapışkan temas bölgelerinde matris moleküllerini yapışkan ile birleştirilir. Mutlaka hücre-matriks arayüzünde güçler dengesini, hızla bir olay alter hücre-matriks yapışmasını değiştiren Ekstrasellüler uyaranlara "dış-iç sinyal" transdüksiyonunda sonuçlanan, mekano duyarlı sinyal proteinleri tarafından 'algılanan'. Bu çapraz-konuşma bahisrinde hücreleri ve kendi çevre hücre dışı matris hücre şekli, motilite, işlev proliferasyonu ve hayatta kalma ² kontrol edilmesinde anahtar bir rol oynar.

Çeşitli pato-fizyolojik süreçler (embriyonik gelişme, iltihaplanma, yara iyileşmesi, kanser metastazı) matris derecelendirme oksidanlar ve / veya enzimler ^3,4 yapışkan matris maddeleri dinamik yeniden ile karakterize edilir. Örneğin, kan damarlarında endotel altı matris proteinleri yapışkan (örneğin, fibronektin) bağlı reaktif oksidanlar lokal üretimi için insan iltihaplı hastalıklarda değiştirilmesi veya bozulması için önemli bir hedef olarak implike edilmiştir (örneğin, hipokloröz asit, HOCl) lökositten türetilmiş bir enzimle inflamatuar damar hastalığı (Şekil 1) ^5-9 esnasında subendotelyuma içinde birikir miyeloperoksidaz (MPO),. MPO türetilmiş oksidanlar ve diğer matris-modifiye, uyarıcıya tepki olarak hücre-matris yapışma değişiklikler lik olanda endotel fonksiyonu ve bariyer bütünlüğünü bozarak endotel hücre sinyal, morfolojisi ve canlılığı, değiştirerek, örneğin; ely patolojik süreçlerin çeşitli sırasında vasküler homeostazı değiştirerek önemli roller oynamaya. Ancak, hücre dışı matriks değişiklikler yapışık hücrelerin morfolojik ve hücre sinyal tepkiler sadece anlaşılmaya başlıyor.

Matris değişiklikler hücre yapışması dinamikleri ve yapışma-bağımlı hücre sinyal yolları değişiklikleri sürücü anlamak için, teknikler doğru yüksek zamansal çözünürlüğe sahip, gerçek zamanlı hücre-matriks yapışma değişiklikleri ölçmek olduğunu gereklidir. Burada, biz tamamlayıcı hücre-substrat empedansını ve bu kriterleri yerine getirmek ve non-invaziv bir şekilde hücre yapışmasını ve de-yapışma süreçleri ölçmek için bir platform sağlamak canlı hücre görüntüleme teknikleri tarif.

Biz göstermek nasıl bu hücre-substrat empedans ve canlı hücre görüntüleme beğenmeağrıları hali hazırda hücre eki dinamiklerini izlemek ve doğal ve modifiye matris substratlar üzerine (örneğin, de novo hücre yapışması) yayılma ve (ii) (örneğin, de-yapışma hücre-matris ayrılma dinamiklerini ölçmek için (i) için kullanılabilir ) matris-modifiye uyaranlara maruz yapışık hücreler tarafından. XCELLigence hücre alt-tabaka empedans biyosensör sistemi 96 oyuklu altın mikroelektrot dizileri yüzeyinde elektrik empedansı ölçmek suretiyle hücre-matris temas alanının bir sürekli ölçme, "hücre dizini ', boyutsuz bir değerdir ve bu elektrik empedans ölçümleri ifade eden Ayrıca (yalıtım) hücre zarı ve elektrot yüzeyi ¹¹ arasındaki ortalama mesafe değişikliklere duyarlı varlık iken, hücre-substrat teması ¹⁰ (Şekil 2) alanına büyük ölçüde orantılıdır. Hücre indeksi değerleri bakımından bir başka artış da sıkı hücre-hücre temas tha oluşması ile elde edilirt, bu çalışmada açıklanan deneyler içinde geçerli değil ¹¹ koşullarını paraselüler akım akar kısıtlamak. Hızlandırılmış diferansiyel girişim kontrast resim analizi ile, zaman içinde hücre çıkıntı yapan alanının ölçümü (DIC), filmler hücre alt-tabaka temas alanında değişiklik tamamlayıcı ölçümünü sağlar ve kesin yapısı ve dinamikleri ile ilgili ek bilgiler sağlar Hücre-substrat empedans yaklaşımı ile ölçülebilir morfolojik değişimler.

Özellikle, (örneğin, fibronektin): (i) yapıştırıcı, alt endotelial matris proteinlerinin oksidasyon MPO aracılı nasıl kullanıldığını izlemek arıtıldı fibronektin ve (ii) tetikler hücre-matris de üzerine süspansiyon endotel hücreleri de novo yapışmasını azaltır, bu yaklaşımların bir uygulama tarif fibronektin kurulan yapışması ile endotel hücreleri -yapışma. Paralel hücre sinyal gerçekleştiren ilgili Biochem kullanarak zamanla analizleri ilenik deneyler (örneğin, Western blot), yapışma bağımlı hücre sinyal olayları adezyon / de-yapışma süreçleri ve ilgili değişiklikleri arasındaki zamansal ve nedensel ilişki belirlenebilir.

Bu yaklaşımlar en son MPO subendotelyal yatakları ile katalize hücre dışı matris oksidasyon önceden var olan aktomizin kasılma güçleri ⁹ tarafından tahrik edilen endotel hücrelerinin hücre-matris yapışma hızlı kayıp tetikler olduğunu göstermek için kullanılmıştır. Önemlisi, hem hücre yapışması değişiklikler ve tespit edilecek sinyal yapışma bağımlı hücre arasındaki zamansal ilişki sağlayarak, bu yaklaşımlar MPO kaynaklı matris modifikasyonu ve hücresel de-yapışma Src kinaz gibi önemli yapışma-bağımlı hücre sinyal yolları değişiklikleri tetikler olduğu tespit bağımlı paxillin fosforilasyon ve miyosin hafif zincir II fosforilleme (Şekil 1) ^9. Redoks-bağımlı sinyal, inv Bu modhücre-matris yapışmasını bozan hücre dışı oksidatif reaksiyonları ile hücre içi sinyal olaylarının aktivasyonu SORUN hücrenin yeni bir şekli "dışarıdan içeriye redoks sinyal" (Şekil 1) olarak adlandırılan sinyalizasyon temsil ^9.

Genel olarak, bu tamamlayıcı hücre alt-tabaka empedans biyosensör ve canlı hücre görüntüleme teknikleri matris düzenleyici uyarı ya da maddeler, hücre yapışması dinamiği, morfoloji değişiklikleri sürücü kadar farklı ifşa ve çeşitli tabi olan farklı yapışık hücre türleri içinde sinyalizasyon değerli olmalıdır Deneysel ayarları.

Aşağıdaki protokol de novo endotel hücre yapışması (Deney 1) ve endotel hücre de-yapışma (Deney 2) süreçleri üzerinde MPO-aracılı matris oksidasyon etkisini ölçmek açıklamaktadır. MPO fibronektin ve diğer yapıştırıcı subendotelyal hücre dışı matriks proteinleri ve bize heyecanla bağlanıres _{(H2O 2)} klorid iyonu (Cı ^-) dönüştürmek için hidrojen peroksit, bu matris proteinleri ile lokal olarak reaksiyona girer ve hücre yapışma özelliklerini (Şekil 1) ^8,9 bozan yüksek reaktif bir klorlama oksitleyici hipokloröz asit (HOCI) ^{'e, 12.}

1. General Endothelial Cell Culture

1. Culture bovine aortic endothelial cells (passages 4–9) on gelatin-coated tissue culture flasks (coat tissue culture surface with 0.1% w/v gelatin in PBS at RT for 15 min) in EGM-2 media (with the EGM-2 bullet kit containing 5% fetal bovine serum, growth factors and all supplements provided by the manufacturer, except for hydrocortisone).
2. When cells are near-confluent (ca. 3 days post seeding after a 1:4 split), harvest cells by treatment with 0.05% w/v trypsin / 0.02% w/v EDTA in PBS at 37 °C. After the majority of cells have detached, add complete EGM-2 media to quench the trypsin and then centrifuge (100 x g, 5 min).
3. Prepare cells for studies on the de novo adhesion of endothelial cells to fibronectin (Experiment 1: Section 2) and the subsequent de-adhesion of endothelial cells with established adhesion on this substrate (Experiment 2: Section 3).
   1. Wash harvested cells once with serum-free Medium 199 containing 1% w/v bovine serum albumin (BSA) and re-centrifuge (100 x g, 5 min).
   2. Re-suspend cells in serum-free Medium 199 containing 1% w/v BSA at 2.5 x 10⁵ cells/ml (cell-substrate impedance measurements) or 5 x 10⁵ cells/ml (live cell imaging analyses) and maintain at 37 °C prior to use.
      NOTE: The adhesion responses of cells are highly sensitive to temperature differences (e.g., due to convection effects) so all equipment and solutions used to handle and treat cells during the following protocols should be kept at a constant temperature of 37 °C.

2. Experiment 1: Quantifying De Novo Endothelial Cell Adhesion on Native and MPO-oxidized Fibronectin (Cell-substrate Impedance)

NOTE: Experiment 1 examines the degree to which MPO-mediated oxidation of fibronectin impairs its ability to support de novo adhesion of suspended endothelial cells.

1. Coat fibronectin onto 96-well gold cell-substrate impedance microelectrode arrays. Add 80 μl/well of purified bovine fibronectin at 5 μg/ml in PBS, incubate for 2 hr at 37 °C and remove the solution.
2. Incubate fibronectin-coated surfaces with MPO to allow the binding of MPO to the surface bound fibronectin. Add 80 μl/well of purified human neutrophil MPO at 20 nM in Hank’s balanced salt solution (HBSS) and incubate for 0.5 hr at 37 °C.
3. Wash surfaces twice with HBSS to remove any unbound MPO.
4. Add H₂O₂ (0-10 μM final concentration) to wells of the microelectrode array plate containing 80 μl/well HBSS to initiate MPO-catalyzed, HOCl-dependent fibronectin oxidation and incubate for another 0.5 hr at 37 °C.
5. To examine the effect of relevant inhibitors or modulators of MPO-catalyzed reactions (e.g., alternative MPO enzyme substrates, enzyme inhibitors or antioxidants; see⁹ for details), add these to the HBSS immediately prior to H₂O₂ addition.
6. After 0.5 hr, treat surfaces with methionine to quench residual surface-bound oxidizing species; i.e., reactive protein-bound chloramines, which may exert confounding cellular activities. Add 10 mM methionine in 80 μl HBSS per well and incubate for 10 min at 37 °C.
7. Block surfaces with BSA. Add 80 μl/well of BSA at 0.2% w/v in PBS, incubate for 2 hr at 37 °C and remove the solution.
   NOTE: Residual uncoated surface regions will support cell adhesion and blocking these with a non-adhesive protein (i.e., BSA) ensures that cellular adhesion responses strictly depend on the purified cell-adhesive matrix employed, in this case fibronectin.
8. Wash surfaces twice with HBSS.
   NOTE: None of the preceding surface treatments appreciably affects cell-substrate readings.
9. Seed suspended endothelial cells (add 200 μl/well at ca. 2.5 x 10⁵ cells/ml, prepared in serum-free Medium 199 containing 1% BSA; see 1.3) onto the native or MPO-oxidized fibronectin coated surfaces.
10. Immediately after seeding cells (i.e., prior to any cell attachment and spreading), mount the microelectrode array plate onto the incubator port (housed in a 37 °C incubator in the presence of 5% CO₂).
    1. Using the instrument software immediately take a ‘blank’ reading to normalize subsequent cell-substrate impedance (‘cell index’) values to the initial background values obtained in the absence of cell-adhesion.
    2. Initiate acquisition of continuous cell index data (minimum of one cell index reading/min).
11. Incubate cells at 37 °C and 5% CO₂ for 2 hr, a time period during which maximal cell attachment and spreading is achieved; this is reflected by a plateauing of the cell index values (see Figure 3A).
    NOTE: The preceding experiment (Experiment 1) examines how initial MPO-mediated oxidation of fibronectin limits the ability of endothelial cells to establish cell adhesion on this substrate. The following experiment (Experiment 2) examines how MPO-mediated fibronectin oxidation promotes decreases in cell-matrix adhesion (i.e., de-adhesion) in endothelial cells with established adhesion onto this substrate. The treatments in these two experiments are essentially identical, except for the timing of MPO-mediated fibronectin oxidation (i.e., before cell adhesion – Experiment 1; after cell adhesion – Experiment 2).

3. Experiment 2: Quantifying Endothelial Cell De-adhesion from Fibronectin in Response to MPO-mediated Fibronectin Oxidation (Cell-substrate Impedance and Live Cell Imaging)

1. Coat fibronectin onto 96-well gold microelectrode arrays for cell-substrate impedance measurements (add 80 μl/well of fibronectin at 5 μg/ml in PBS and incubate for 2 hr at 37 °C) or 35 mm glass-bottomed cell culture dishes for live cell imaging analyses (add 2 ml/dish of fibronectin at 5 μg/ml in PBS and incubate for 2 hr at 37 °C).
2. Block surfaces with BSA. Add BSA at 0.2% w/v in PBS at the volumes indicated in 3.1 and incubate for 2 hr at 37 °C.
3. Incubate surfaces with MPO to allow the binding of MPO to fibronectin. Add 20 nM purified human MPO in HBSS at the volumes indicated in 3.1 and incubate for 0.5 hr at 37 °C.
4. Wash surfaces twice with HBSS to remove any unbound MPO.
5. Seed suspended endothelial cells (prepared in serum-free Medium 199 containing 1% w/v BSA; see 1.3) onto the native or MPO-bearing fibronectin coated surfaces.
   1. Add 200 μl/well at 2.5 x 10⁵ cells/ml to 96 well cell-substrate impedance microelectrode arrays.
   2. Add 2 ml/dish at 5 x 10⁵ cells/ml to 35 mm glass bottomed cell culture dishes.
6. Immediately after seeding cells (i.e., prior to any cell attachment and spreading):
   1. Mount 96 well microelectrode array plates onto the cell-substrate impedance incubator port (housed in a 37 °C incubator in the presence of 5% CO₂) and take ‘blank’ readings to initiate continuous acquisition of cell index data (cf. Section 2.10).
   2. Transfer 35 mm glass bottomed cell culture dishes to a 37 °C incubator in the presence of 5% CO₂.
7. Incubate cells at 37 °C for 2 hr to allow maximal cell attachment and spreading (cf. Section 2.11).
8. Briefly remove the 96 well microelectrode array plate (pause cell index readings at this point) or 35 mm glass bottomed cell culture dish from the 37 °C incubator, remove cell supernatant and add warmed (37 °C) HBSS (volumes as per step 3.1).
   NOTE: HBSS is employed when studying MPO-catalyzed oxidative reactions instead of complete culture media as the latter contains oxidizable species that interfere with the oxidation reactions.
9. To examine the effect of inhibitors/modulators of MPO-catalyzed reactions (alternative enzyme substrates, enzyme inhibitors or antioxidants) or cell signaling inhibitors (e.g., 40 μM blebbistatin to inhibit actomyosin contractility), add these now.
10. Immediately place the microelectrode array plate back into the 37 °C incubator port (re-commence cell index readings at this point) or the 35 mm glass bottomed cell culture dish back into the 37 °C incubator, and allow the cells to equilibrate in the HBSS for 0.5 hr.
    NOTE: After 0.5 hr equilibration in HBSS, cell index values stabilize at slightly lower values; when pre-incubating cells with enzyme substrates, enzyme and cell signaling inhibitors or antioxidants, first ensure that these do not significantly affect the values obtained after equilibration.
11. Initiate MPO-catalyzed, HOCl-dependent fibronectin oxidation and de-adhesion.
    1. For cell impedance studies using 96 well microelectrode array plates:
       1. Remove the microelectrode array plate from the incubator and pause cell index measurements.
       2. Add H₂O₂ (final concentration of 0-10 μM) to the HBSS and gently mix by repeated pipetting.
       3. Immediately, re-mount the microelectrode array back onto the 37 °C incubator port and re-commence the acquisition of cell index readings.
    2. For live cell imaging studies using 35 mm glass bottomed cell culture dish:
       1. Remove the culture dish from the incubator and mount onto a 37 °C heated stage of an inverted confocal microscope equipped with a 63X water objective lens with suitable DIC optics for recording live cell movies.
       2. Focus on cells and optimize DIC optics (Köhler illumination, bias retardation and camera offset/gain; for details, see¹³).
       3. Initiate DIC movie and record baseline readings of untreated cells for 1 min.
       4. Add H₂O₂ (final concentration of 0-10 μM) and gently mix by repeated pipetting. Re-focus the microscope (if necessary) and continue recording DIC movies of the treated cells for the required time period.

4. Data Analysis and Presentation

1. Cell-substrate impedance data
   1. Export raw data (cell index versus time) into a spreadsheet.
   2. For cell de-adhesion studies (Experiment 2: Section 3), normalize data by setting values recorded immediately prior to the initiation of MPO-mediated fibronectin oxidation at a value of 1 (i.e., immediately before the addition of H₂O₂ in 3.11.1.1).
      NOTE: This ensures that relative changes in cell index values elicited by MPO-mediated fibronectin oxidation are not masked by small initial differences in absolute cell index values between wells.
      1. Present data as plots of normalized cell index (y-axis) versus time (x-axis).
2. Live cell imaging data (cell de-adhesion studies in Experiment 2: Section 3)
   1. Open DIC live cell imaging movies recorded immediately prior to and after the initiation of MPO-mediated fibronectin oxidation in a standard image analysis program (e.g., ImageJ software).
   2. In at least two separate DIC movies randomly select multiple cells and measure their projected area in sequential frames (e.g., at 1 min intervals) by manually tracing their membrane edge and quantifying the number of enclosed pixels.
   3. Export raw data (projected cell area versus time) to an Excel spreadsheet and normalize cell area data by setting values recorded immediately prior to the initiation of MPO-mediated fibronectin oxidation at a value of 1 (i.e., immediately before the addition of H₂O₂ in 3.11.2.3).
   4. Present data as a plot of normalized cell area (y-axis) versus time (x-axis).

Real-time quantification of endothelial cell de-adhesion from fibronectin in response to MPO-mediated fibronectin oxidation (Experiment 2). The seeding of endothelial cell suspensions onto native (MPO free) fibronectin or MPO-bearing fibronectin results in maximal cell attachment and spreading within 2 hr, as judged by a plateauing of cell index values in the cell substrate impedance measurements (Figure 3A). This initial phase of cell attachment and spreading is markedly reduced when MPO-mediated fibronectin oxidation is initiated prior to cell seeding in experiments performed according to the protocol detailed in Experiment 1 (data not shown; For details see⁹). Once maximal cell adhesion is established on native or MPO-bearing fibronectin, targeted fibronectin oxidation mediated by MPO-catalyzed HOCl (initiated by the addition of MPO’s co-substrate H₂O₂) causes a rapid decrease in the surface area of cell-matrix contact measured by cell-substrate impedance (Figure 3A,B) and by live cell imaging (Figure 3C; for a representative DIC movie, see Movie 1; cell index or cell area losses after H₂O₂ addition are minimal in the absence of MPO⁹). Whilst cell index and cell area changes during MPO-induced de-adhesion were very similar, the comparatively slower decreases in cell index values may reflect the insulating effects of cell membrane materials present in ‘cell-free’ regions at the cell periphery during de-adhesion, which are not quantified in the projected cell area measurements. The rapid cellular de-adhesion apparent in endothelial cells bound to MPO-bearing fibronectin in response to H₂O₂ treatment is absent in cells treated with H₂O₂ alone (i.e., cells that do not contain MPO) (Figure 3A) and is blocked by MPO enzyme inhibitors or HOCl scavengers (data not shown; see⁹ for details), identifying that this process is dependent on the MPO-catalyzed production of HOCl (cf. Figure 1). Inhibition of myosin II motor function with blebbistatin inhibits the rate of endothelial cell de-adhesion measured by cell-substrate impedance (Figure 3B) and by live cell imaging (Figure 3C, Movie 2), identifying that cellular de-adhesion and contraction in response to MPO-catalyzed subcellular matrix oxidation is driven by actomyosin tensile forces⁹.

Figure 1. MPO-mediated subcellular matrix oxidation triggers cell-matrix de-adhesion and cell signaling. MPO triggers rapid de-adhesion responses and changes in adhesion-dependent signaling by mediating targeted oxidation of adhesive subendothelial matrix proteins, involving the following events⁹: (A) MPO binds avidly to the subendothelial matrix and uses H₂O₂ to generate the highly reactive oxidant HOCl that reacts locally with matrix proteins (e.g., fibronectin) and disrupts their cell adhesive properties. (B) This damage disrupts adhesive contacts at the cell-matrix interface, leading to (C) membrane retraction driven by unopposed tension in the actin cytoskeleton and the alteration of adhesion-dependent cell signaling pathways (Reproduced with permission from Rees et al.⁹). Please click here to view a larger version of this figure.

Figure 2. Work-flow and principle of cell-substrate impedance analyses to quantify endothelial cell de-adhesion from fibronectin in response to MPO-mediated fibronectin oxidation (Experiment 2). Cell index values measured by the cell-substrate microarray impedance biosensor system reflect the ability of the cell membrane to limit field-induced ion currents at the electrode surface and are proportional to the area of cell–substrate contact¹⁰. Please click here to view a larger version of this figure.

Figure 3. Real-time quantification of endothelial cell de-adhesion from fibronectin in response to MPO-mediated fibronectin oxidation (Experiment 2). Endothelial cell suspensions (in serum-free Medium 199 containing 1% BSA) were seeded onto native or MPO-bearing fibronectin (fibronectin coated at 5 g/ml, then incubated in the absence or presence of 20 nM MPO) in 96 well microelectrode arrays (cell-substrate impedance measurements) or 35 mm glass-bottom cell culture dishes (live cell imaging analyses). Cells were then incubated at 37°C for 2 hr to allow maximal cell attachment and spreading before equilibrating cells with HBSS and initiating MPO-mediated fibronectin oxidation by adding H₂O₂ (10 μM) (time of H₂O₂ addition set at t = 0 min). (A) Full time-course of cell-substrate impedance measurements before and after treatment with H₂O₂ in the presence (+MPO) and absence (-MPO) of MPO. (B) Cell-substrate impedance measurements and (C) cell area measurements by live cell imaging analyses, after treatment of MPO-containing cells with H₂O₂ in the presence (+ blebbistatin) and absence (-blebbistatin) of the myosin II inhibitor blebbistatin (40 μM). Cell index and cell area values are normalized to values immediately prior to the time of H₂O₂ addition, which were given a value of 1. Cell index data represent the mean SEM, n = 6 replicate measurements from a representative experiment. Cell area values represent the mean SEM, n = 10 cells (2 replicate movies, 5 randomly selected cells per movie: see Movies 1 and 2). (Figure 3B, Figure 3C, Movie 1 and Movie 2 are reproduced with permission from Rees et al.⁹). Please click here to view a larger version of this figure.

Movie 1. Time lapse DIC microscopy movie of endothelial cells adhered on MPO-bearing fibronectin over 0 – 15 min following exposure to H₂O₂ (10 μM); 1 sec = 3 min.

Movie 2. Time lapse DIC microscopy movie of endothelial cells adhered on MPO-bearing fibronectin over 0 – 15 min following exposure to H₂O₂ (10 μM) in the presence of blebbistatin (40 μM); 1 sec = 3 min.

Cell-matrix adhesion and de-adhesion processes can be quantified accurately in real time with high temporal resolution using cell-substrate impedance and live cell imaging analyses. These real-time approaches provide a major advantage over end-point analyses of cell adhesion, which provide poor temporal resolution. By accurately quantifying rapid de-adhesion responses with high temporal resolution, these analyses can provide critical insights into how morphological responses to matrix modifications are regulated and how they impact on adhesion-dependent cell signaling processes, measured in parallel using relevant biochemical assays (e.g., Western blotting).

In recent studies, these approaches were used to reveal a novel role for MPO-mediated oxidation of the subendothelial matrix in triggering the de-adhesion of endothelial cells and showed: (i) that the rate of de-adhesion was critically dependent on pre-existing actomyosin contractility (cf. Figures 3B and 3C) and (ii) that the loss of cell-matrix adhesion drove changes in important adhesion-dependent cell signaling pathways including Src-dependent paxillin phosphorylation and myosin light chain II phosphorylation⁹ (see Figure 1). These data have important implications for understanding endothelial function and barrier integrity during inflammatory responses, where the subendothelial extracellular matrix is implicated as a key target for oxidants produced by subendothelial deposits of matrix-bound MPO or other sources of reactive oxidants ^3,5-9,14.

The use of a microarray-based, cell-substrate impedance biosensor system provides a platform for robust, high-throughput cell adhesion measurements. Each well of the 96 well cell-substrate impedance microelectrode arrays has the potential to simultaneously analyze 32 different experimental conditions (i.e., 3 wells per condition). However, when comparing small changes in cell adhesion, at least 4 or more wells should be used per experimental condition. During cell seeding and subsequent cell treatments, care should be taken to keep all solutions at 37 °C and minimize the time taken to treat cells outside the 37 °C incubator environment (this avoids potential temperature differences across the microelectrode array that may affect adhesion responses). Notably, the electrical current at the cell-substrate interface is sensitive to the ionic strength of the cell supernatant¹⁰: consequently, cells should be allowed to equilibrate after the addition of new buffers/media to obtain a steady cell index response before monitoring the effect of the stimulus of interest (see 3.10 and Figure 3A). Decreases in cell index may not only reflect a decrease in the average area of cell-matrix contact but may also reflect a decrease in the number of attached cells. To discriminate between these possibilities, changes in the number of attached cells on microelectrode arrays can be quantified immediately after cell-substrate impedance measurements by Crystal violet staining this approach has been used to confirm that MPO-mediated fibronectin oxidation triggers a rapid decrease in the average area of cell-matrix contact, but does not promote cell detachment (for details, see⁹).

A limitation of the cell-substrate impedance technique is that it provides an average measure of adhesion changes over all cells present on the microelectrode surface and it does not give information about the precise nature of adhesion or morphological changes at the level of individual cells. To address this limitation, live cell DIC microscopy and image analysis provides a complementary measure of adhesion changes and reveals morphological changes of individual cells. Thus, decreases in cell index in response to MPO-mediated fibronectin oxidation measured by cell-substrate impedance (Figure 3A) correlate well with the decreases in the projected area of cells measured by live cell image analysis of DIC movies (Figure 3B). Importantly, DIC movies reveal that, in individual cells, MPO-induced de-adhesion involves the rapid retraction of the peripheral cell membrane from the substratum and adjacent cells and ultimately results in cells assuming a compact ‘rounded-up’ morphology, but without resulting in cell detachment (Movie 1). As with cell-substrate impedance measurements, to ensure reproducible cellular responses, care should be taken to maintain solutions at 37 °C prior to use on cells and a heated microscope stage (or equivalent temperature control device) must be employed during the live cell imaging recordings.

The protocols described here can be modified readily by substituting fibronectin with other purified matrix substrates (e.g., collagen or laminin) or studying a more complex matrix environment produced overtime by cultured adherent cells (for details, see⁹). The protocols can also be altered by substituting MPO-mediated matrix oxidation with other soluble stimuli that disrupt cell-matrix adhesion including other physiological matrix-modifying oxidants (e.g., peroxynitrite, nitrogen dioxide radical^5-8), matrix-degrading proteases¹⁵ or antagonists of adhesive ligands present on the cell surface or in the matrix (e.g., anti-integrin antibodies or RGD peptides). Live cell imaging analyses can also be used to quantify cell-matrix de-adhesion in response to the electrochemical desorption of cell-matrix contacts¹⁶. Finally, the described protocols are also readily applicable to studying cellular adhesion dynamics in adherent cells other than endothelial cells (e.g., epithelial cells).

The authors have no disclosures to make.

This work was funded by a National Health and Medical Research Council (NHMRC) Project Grant 568721 (SRT) and, in part, by a UNSW Faculty of Medicine Faculty Research Grant (MDR).