Microscopy and image analysis of individual and group cell shape changes during apoptosis

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1 A. Méndez-Vilas and J. Díaz (Eds.) Microscopy and image analysis of individual and group cell shape changes during apoptosis Mark A. DeCoster*, Sowgandhi Maddi, Vivek Dutta and James McNamara Biomedical Engineering Program and Institute for Micromanufacturing, Louisiana Tech University, Ruston, Louisiana, USA, *Corresponding Author We have previously described the nuclear area factor (NAF) as one indicator of cell shape changes associated with apoptosis. NAF changes during apoptosis would be expected to be most dramatic for highly morphologically polarized cells such as brain cells. To test this we carried out analysis of NAF changes in Jurkat cells, which are normally round in shape. We found that while the average NAF changed little after stimulation of apoptosis with staurosporine in Jurkat cells, nuclear staining with DAPI and activated caspase-3 both increased. We also discovered that prior to the changes noted above, intercellular group cell morphology changed as expressed by cell clumping. Clumping was assessed by image analysis of calcein-stained cells as well as from phase microscopy images of the same fields. Analysis of cell size, shape, and expression of apoptosis-associated markers is shown for both individual and clumped cells, including methods for analysis of digital microscopy images for these data. We used image processing algorithms to verify the morphological changes induced during the process of apoptosis in the Jurkat cells. The algorithms constitute image denoising mechanisms, image segmentation and contour extraction methods before we derived the data on parameters such as cell- and clump-size. The digital images were analyzed in Matlab and Image-Pro software platforms and the results were compared to theoretical expectations of results. Apoptosis-induced changes in normally round Jurkat cells were also compared with those observed in morphologically polarized brain cells. Determining the timing of morphological and biochemical changes associated with apoptosis for different cell types will be important for understanding the discrete steps of cell death. Keywords apoptosis; nuclear area factor (NAF); image analysis; cell shape; Jurkat cells; brain cells 1. Introduction Inter- and intra-cellular changes as recorded by modern microscopy are revealing much information about how cells behave normally and in disease. In addition, this insight is important for our understanding of defining environments for controlled cell growth and cell assemblages. As a recent example, Brandy et al. have demonstrated directed assembly of yeast cells into highly spherical cell aggregates by microbubble templating [1]. In the current review, we report a novel transition of Jurkat cells into highly spherical structures during apoptosis induced by staurosporine. As we have previously described [2,3], the nuclear area factor (NAF) may be used as one descriptor for apoptosis. Since Jurkat cells are normally round, their transition to an apoptotic state includes shrinkage in cell size without a major change in cell shape. This is in contrast to other cell types used here, which include normal brain cells. 2. Materials and Methods 2.1 Cell culture Jurkat cells were obtained from and grown according to vendor recommendations (American Type Culture Collection, Manassas, VA) in RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. Brain hippocampal astrocytes were obtained from Lonza and grown in Kaighn s F-12 Ham modified basal media supplemented with 5% each of fetal bovine and horse serum, and penicillin/streptomycin. All cells were maintained at 37 C in a humidified incubator containing 5% CO Cell staining and Microscopy After apoptotic stimulus, cell nuclei were stained with 4,6-Diamidino-2-phenylindole dihydrochloride (DAPI) and examined with a Nikon inverted fluorescence microscope as previously described [2]. A key aspect of this technique is that cells remain alive during this procedure, reflecting cells with natural morphology as opposed to changes in shape which may occur due to fixation. Calcein staining also is carried out on living cells, and was utilized here to determine the area of individual and grouped cells using procedures as we have recently described [4]. Five microliters of pluronic acid at a concentration of 20% (weight/volume) in DMSO and 10 µl of a stock solution of calcein AM at a concentration of 1 mm were added into 5 ml of pre-warmed Locke s solution which had been equilibrated to 37 C. This loading solution was added to 836

2 A. Méndez-Vilas and J. Díaz (Eds.) cells for 25 minutes in a CO 2 incubator, after which cells were imaged with a Nikon inverted fluorescence microscope using a 485 nm excitation filter and a monochrome camera. Calcein AM is a vital fluorescent dye, in that it only fluoresces once inside the live cell. The fluorescent moiety is conjugated to an acetyl methyl ester, which aids in loading the living cells and blocks the fluorescence until the ester is cleaved by naturally occurring esterases inside the cell. In this manner the cleaved dye is also trapped within the cell after esterase action. Once cells are loaded with the dye, living cells fluoresce brightly. With little or no fluorescence outside the cells, images can be made with very good detail, and little or no background noise. Fluorescence microscopy is known for its potentially high signal to noise ratio, and calcein staining is an excellent example, as some other fluorophores must be washed out of the sample due to the fact that they are intrinsically fluorescent. Calcein AM loading allows for specific fluorescent labeling within living cells. 2.3 Biochemical determination of Apoptosis: Caspase-3 Assay. Biochemical determination of apoptosis was measured using a CASP3-C kit from Sigma Aldrich to determine caspase- 3 activity, which was compared to a p-nitroaniline standard and the specific Ac-DEVD-pNA substrate for the enzyme. Specificity of the reaction was further verified using a specific caspase inhibitor (Ac-DEVD-CHO) provided in the kit. Cell lysates were prepared from apoptotic cell cultures at the indicated times shown in results and the final reaction product measured at 405 nm using a Thermo Scientific Multiskan Spectrum plate reader. 2.4 Image processing using Adobe Photoshop, Image Pro Plus, and Matlab software Added images for combining phase and fluorescence fields utilizing calcein and DAPI staining (Figures 3 and 9, respectively), were created using Adobe Photoshop CS, version 8.0. Fluorescent (calcein-stained) images were chosen as the source and added (using the Add blending function under Apply Image tools) to the corresponding phase microscopy field, which was chosen as the target image. Opacity was set at 75% for all added images. The Apply Image tools are found under the Image tab in this version of Photoshop. Analysis of the number of calcein- and DAPI-stained images after induction of apoptosis was determined using Image Pro Plus software (version 6.1). Objects were defined by choosing the count bright objects function, which identified individual and grouped cells stained with calcein and DAPI. The clean borders function was used to avoid counting cells touching or cut by the edged of the captured field. The split objects function was used to separate cells touching each other. This especially presents a problem in calcein-stained astrocytes, depending on the cell density. Most DAPI-stained nuclei in cell culture do not touch. NAF was calculated as we have previously described [2,3]. In general, since cell nuclei become more round and smaller in size during apoptosis [2,3], the NAF decreases in value during apoptosis. Here, NAF was reported in square pixels (see Figure 10A). Cell area factor (CAF) was calculated by multiplying the area of the calcein stained object times the roundness, resulting in an area in square pixels (Figure 10B). In Image pro plus ver 6.1 software, the formula for roundness is (perimeter2)/(4*pi*area), with 1.0 indicating a perfect circle and larger values indicating oblong and non-circular objects. Analyses of digitized microscopy images for calcein using MATLAB software were based on an algorithm with manual thresholding (MATLAB version R2009b). To calculate the average area of objects for each time point, the number of pixels within all contours was calculated and the average of the number of pixels within each contour was calculated. Steps in the image processing are shown in Figure 6, and resulting data analysis shown in Figure 7 (right panel). Analyses of digitized microscopy images for DAPI staining using MATLAB software were carried out using an algorithm by manually thresholding to partition the nuclei and DNA fragments (appearing bright) and to separate these identified objects from the rest of the background (appearing dark). A threshold value close to a peak in the histogram near the brighter end of the scale was chosen to partition the image. The partitioned image was converted to a binary image. Then, the number of bright objects was counted for each image at respective time points. Steps in the image processing for DAPI using MATLAB are shown in Figure 5, and resulting data analysis shown in Figure 7 (left panel). 3. Results and Discussion As a normally round cell derived from peripheral blood, Jurkat cells would not be expected to change dramatically in cell shape (roundness) during apoptosis, while they might be expected to decrease in cell area (line pair B in Figure 1). This is in contrast to brain cells, which are highly morphologically polarized, starting from a very non-round shape, to more round as they undergo apoptosis. In addition the cell area would be expected to decrease during apoptosis as well (line pair A in Figure 1). These relative changes in different cells types are shown in Figure 1, and could be applied to changes in cell area and cell roundness as well as nuclear area and nuclear roundness as we have previously described [2,3]. Therefore, considering cell area and cell roundness changes, we suggest the term cell area factor (CAF), as analogous to the nuclear area factor (NAF) as we have previously discussed [2,3]. 837

3 A. Méndez-Vilas and J. Díaz (Eds.) Figure 1. Schematic representation of relative roundness and area changes for brain cells (line pair A) and blood-derived cells (line pair B) during apoptosis. Note that non-round objects start at higher values, with perfectly round objects having a value of 1. Staurosporine is a well known inducer of apoptosis [5]. Here we used Staurosporine to induce apoptosis in Jurkat cells, and discovered a transitional point at which extensive cell clumping occurred (Figure 2). Figure 2. Transition of Jurkat cell clumping during apoptosis. Cells at an initial density of 75,000 per ml were treated with 2 µm staurosporine in complete growth medium, after which phase microscopy images were obtained at 0, 1, 2, and 4 hours as shown in panels a, b, c, and d, respectively at 200 x magnification. It is interesting to note the transition of Jurkat cells once treated with staurosporine, from individual cells to spherical clumps of cells at intermediate timepoints, then back to individual objects as apoptosis becomes more advanced. Part of this intercellular dynamic may be due to changes in charge on the cell membrane. This concept has a basis in Layer-byLayer (LbL) strategies used for example to encapsulate bacteria as individual units [6], and more recently, for aid in the formation of spherical yeastosomes comprised of multicellular aggregates [1]. Upon discovering this transition of Jurkat cells from individual to clumped formations, we used the well-known vital fluorescent indicator calcein to label cells as we have recently described [4]. Figure 3 shows the digital microscopic images of the observed transition from individual cells to clumped groups of cells, back to individual objects as revealed by calcein staining. Figure 3. Transition of Jurkat cell clumping during apoptosis as indicated by calcein staining. Cells at an initial density of 75,000 per ml were treated with 2 µm staurosporine in complete growth medium, after which phase and fluorescence microscopy images were obtained at 0, 2.5, and 6 hours as shown in panels a, b, and c, respectively at 200 x magnification. Each image is an added image of the same field of cells obtained for phase and fluorescence; added images were created in Abobe Photoshop as described in methods. 838

4 A. Méndez-Vilas and J. Díaz (Eds.) The benefit of the observed changes for tracking apoptosis is two-fold: 1) Calcein staining provides a much better signal to noise ratio than traditional white-light microscopy images, which aids in image analysis for computing identified object parameters such as average object size and shape and 2) since calcein is a vital stain, stained cells and groups of cells indicate early apoptosis rather than necrosis, since cells are still viable; as late apoptosis occurs and cell death progresses, calcein staining would presumably be lost. Thus calcein itself may be a marker for transition points in the cell death process. Indeed, as can be seen in Figure 3, a number of smaller objects lacking calcein staining begin appearing as apoptosis proceeds at 2.5 and 6 hours post-treatment with staurosporine (panels 3b and 3c, respectively). Control cells have few if any objects lacking calcein staining (Figure 3a). To our knowledge, this is the first demonstration and analysis of this transition from individual cells to clumps of cells in Jurkat cells undergoing apoptosis. It is interesting to note that Jewett et al. reported aggregation of peripheral blood mononuclear cells (PBMCs) after addition of an oral bacterium that induced apoptosis in the PBMCs [7]. The authors noted that the aggregation preceded the appearance of apoptosis; however, the data for aggregation were not shown. When we analyzed the number of cells staining for calcein or DAPI over time after induction of apoptosis, we found that while DAPI-positive cells increased linearly over time after induction, the number of calcein-positive objects decreased sharply after induction, followed by a linear rise paralleling DAPI staining kinetics (Figure 4). Figure 4. Transition of Jurkat cell clumping during apoptosis as indicated by calcein and DAPI staining. Cells at an initial density of 75,000 per ml were treated with 2 µm staurosporine in complete growth medium, after which fluorescence microscopy images were obtained at the indicated times for calcein and DAPI staining. The number of bright objects for DAPI staining and live objects for calcein staining were determined using Image Pro Plus software as described in Methods. MATLAB software was also used along with Image Pro Plus to analyze the dynamics of apoptosis from digital microscope images of cells stained with both DAPI and calcein. For DAPI-stained images, an example of MATLAB processing is shown for one timepoint in Figure 5. Figure 5. MATLAB image analysis of apoptosis in Jurkat cells treated with staurosporine: DAPI staining. Left panel: Original fluorescent image of Jurkat cells when treated with staurosporine and stained with DAPI (5 hours post-treatment). Right panel: the resulting partitioned image from the left panel image after MATLAB processing, showing DNA fragments and apoptosis due to staurosporine treatment. Original microscope magnification of images= 200 x. 839

5 A. Méndez-Vilas and J. Díaz (Eds.) MATLAB was also used to analyze the average area of objects stained with calcein during the apoptosis process. A sequence of the MATLAB processing for calcein-stained images is shown in Figure 6. Figure 6. MATLAB image analysis of apoptosis in Jurkat cells treated with staurosporine: Calcein staining. A contrast enhanced fluorescent image of Jurkat cells stained with calcein is shown at time 0 (left panel). Middle panel shows the same image after segmentation using MATLAB. Right panel shows the skeletons or the boundaries of the contours in the middle image obtained using MATLAB. Original microscope magnification of images= 200 x. Analysis of these data sets for DAPI and calcein staining using MATLAB also indicated a linear increase of DNA damage as demonstrated by increased numbers of DAPI-stained nuclei and nuclear fragments with increased time after initiating apoptosis with staurosporine, (Figure 7, left panel). Figure 7. Analysis of apoptosis in Jurkat cells treated with staurosporine: MATLAB image analysis. DNA-fragmentation is indicated by DAPI staining (left panel), and the average area of live objects stained with calcein is shown in right panel). For both figures, variation in two analyzed data sets is shown by triangles, average values are shown by circles and the solid line shows the trendline. This trend was similar to what was calculated using Image Pro Plus software for DAPI-stained cells (Figure 4). For calcein stained cells, as seen in Figure 7 (right panel), the area of identified objects initially increases, as Jurkat cells clump during the early phases of apoptosis (1-2.5 hours), and then area dramatically decreases as cells become more fragmented (3 hours and after). This nicely parallels in an inverse manner the curve of the number of calcein-stained (live) objects as analyzed by Image Pro Plus software (Figure 4). Thus, for calcein-stained Jurkat cells under these conditions, analysis shows that the area of stained objects increases in early phases of apoptosis as cells clump (Figure 7), and since there are a finite number of cells, the number of objects thus decreases during this early phase (Figure 4). In contrast to Jurkat cells, brain cells are highly morphologically polarized and not round in shape. However, to investigate whether cell shape could be used as an indicator for apoptosis-associated changes in brain cells, we loaded cells with calcein and then analyzed cell shape from digital images of untreated brain astrocytes or astrocytes treated with staurosporine for 4 hours (Figure 8). 840

6 A. Méndez-Vilas and J. Díaz (Eds.) Figure 8. Transition of brain astrocyte cell area and shape as indicated by calcein staining. Cells were treated with 2 µm staurosporine in complete growth medium, after which fluorescence microscopy images were obtained at 0 and 4 hours as shown in panels a and b, respectively at 100 x magnification. In addition, to compare nuclear changes for brain cells and Jurkat cells undergoing apoptosis, we loaded brain astrocytes with DAPI and analyzed digital images of these astrocytes either in control (untreated) conditions, or after 5 hours of treatment with staurosporine (Figure 9). Figure 9. Transition of brain astrocyte nuclear area and shape as indicated by DAPI staining. Cells were treated with 2 µm staurosporine in complete growth medium, after which phase and fluorescence microscopy images were obtained at 0 and 5 hours as shown in panels a and b, respectively at 200 x magnification. Each image is an added image of the same field of cells obtained for phase and fluorescence; added images were created in Abobe Photoshop as described in methods. Analysis of apoptosis-associated changes in Jurkat cells and brain astrocytes is shown in Figure 10. For both Jurkat cells and astrocytes, the nuclear area as indicated by DAPI staining decreased with staurosporine treatment; when multiplied by the nuclear roundness, the Nuclear Area Factor (NAF) was calculated, which also decreased upon treatment with staurosporine for both cell types (Fig. 10A). For calcein staining of cells we derived a new morphological indicator, which we call the Cell Area Factor (CAF). Like NAF, the CAF takes into consideration the roundness of the cell, which is multiplied by the cell area. As can be seen in Figure 10B, normal brain astrocytes tend to have a much larger area, and much larger CAF than Jurkat cells, since the astrocytes are not round in the normal state. However, as the cells undergo apoptosis after staurosporine treatment, the area and CAF are highly decreased in astrocytes, forming in some cases cellular fragments which can still be imaged using calcein (Figure 8b). Finally, as shown in Figure 10C, the roundness of Jurkat cells does not dramatically change under early apoptosis conditions (6 hours or less in these studies), when measured either for the nucleus (DAPI) or for the whole cell with calcein. In contrast, whole cell roundness in astrocytes is highly skewed from a circular shape in the starting state, while 841

7 A. Méndez-Vilas and J. Díaz (Eds.) approaching more round during apoptosis as indicated by calcein staining. Astrocyte nuclei start round and become a bit more round during apoptosis (Fig. 10C). Figure 10. Analysis of area, nuclear area factor (NAF), cell area factor (CAF), and roundness in Jurkat cells and astrocytes (astros) during apoptosis. Nuclear (NAF) and cell (CAF) morphological changes were measured in Jurkat cells and astrocytes as indicated in Methods. C= control and +S= addition of staurosporine for 6 hours. Data shown are the average of from cells depending on the condition and standard error bars are indicated. When we measured activated caspase-3 activity in Jurkat cells and astrocytes under these conditions of apoptosis it was interesting to note that caspase-3 activity had not peaked yet at 1 hour (Figure 11), while cellular clumping had begun as indicated by morphological microscopy evaluation (Figure 2b), decrease in the number of live objects (Figure 4), and the increase in average area of these live objects (Figure 7, right panel). As adherent cells, astrocytes are not expected to clump under these conditions, but they show significant caspase-3 activity after staurosporine treatment [8]. Ernest et al. have discussed how cytoplasmic condensation is both necessary and sufficient to induce apoptotic cell death [9] a phenomenon we have measure here in astrocytes by a huge decrease in the cell area and cell area factor (CAF) after staurosporine treatment (Figure 10B). Cytoplasmic condensation is also illustrated by the small, punctuate staining using calcein on astrocytes undergoing apoptosis (Figure 8b). Under the conditions of our experiments, caspase-3 activity in astrocytes appears to parallel the time course of Jurkat cell responses to staurosporine, with higher activity measure in astrocytes compared to Jurkat cells at all time points measured (Figure 11). Combined with biochemical assays, the quantifiable measures of area and roundness as shown schematically in Figure 1, can be applied to track steps in the apoptotic process. Area and roundness for the cell nucleus can be used to calculate a nuclear area factor (NAF), and area and roundness of cellular structure using vital dyes such as calcein may be used to calculate a cell area factor (CAF). Figure 11. Activated caspase-3 activity in Astrocytes and Jurkat cells after treatment with staurosporine. Caspase-3 activity was assayed as described in Methods. Data shown are representative from 3 separate experiments for each condition on each cell type. Acknowledgements This work was supported in part by the Louisiana Board of Regents PKSFI Contract No. LEQSF ( )ENH-PKSFI-PRS-04 and NSF Award #

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