Anal. Chem. 2008, 80, 9349–9354
Construction of Time-Lapse Scanning Electrochemical Microscopy with Temperature Control and Its Application To Evaluate the Preservation Effects of Antifreeze Proteins on Living Cells Yu Hirano,* Yoshiyuki Nishimiya, Keiko Kowata, Fumio Mizutani,† Sakae Tsuda, and Yasuo Komatsu* Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira, Sapporo, Japan Antifreeze proteins (AFPs) can protect cells from hypothermic damage; however, their mechanism of action remains unclear. Scanning electrochemical microscopy (SECM) can evaluate the size and activities of cells, although long-term continuous monitoring has been unsuccessful. We constructed a novel, fully automated, timelapse SECM system and investigated the cell preservation effect of AFPs by analyzing single cellular topography at low temperatures. From the SECM measurements, mammalian cells (HepG2), treated in Euro-Collins (EC) solution at 4 °C, began to swell at 8 h and then immediately ruptured. In AFP-containing EC solution, the cellular size did not change until 16 h and then gradually increased and finally ruptured. In addition, the cellular height at rupture point significantly increased in the presence of AFPs. These results suggest that AFPs stabilize the cellular membrane and protect cells from hypothermic damage. This SECM system allowed us to observe the single cellular response to hypothermia by long-term automatic scanning and will be applicable for analysis to other cellular activities and topographies. Antifreeze proteins (AFPs) have the unique ability to bind to the surface of ice crystals. This ice-binding ability of AFPs provides a decrease in freezing temperature and an inhibition of ice recrystallization.1-4 In addition, AFPs have been suggested for use in protecting the cell and organ function from hypothermic damage.5-7 Hypothermic preservation is extensively used for the * To whom correspondence should be addressed. E-mail:
[email protected] (Y.H.) and
[email protected] (Y.K.). Phone: +81-11-857-8523. Fax: +8111-857-8954. † Current address: Graduate School of Material Science, University of Hyogo, 3-2-1 Kouto, Kamigori-cho, Ako-gun, Hyogo, Japan. (1) Knight, C. A.; Cheng, C. C.; Devries, A. L. Biophys. J. 1991, 59, 409–418. (2) Yeh, Y.; Feeney, R. E. Chem. Rev. 1996, 96, 601–617. (3) Harding, M. M.; Ward, L. G.; Haymet, A. D. J. Eur. J. Biochem. 1999, 264, 653–665. (4) Davies, P. L.; Sykes, B. D. Curr. Opin. Struct. Biol. 1997, 7, 828–834. (5) Wang, J. H. Cryobiology 2000, 41, 1–9. (6) Inglis, S. R.; Turner, J. J.; Harding, M. M. Curr. Protein Pept. Sci. 2006, 7, 509–522. (7) Rubinsky, B. Heart Failure Rev. 2003, 8, 277–284. 10.1021/ac8018334 CCC: $40.75 2008 American Chemical Society Published on Web 11/04/2008
preservation of whole organs and is beneficial for prolonging the viability of living cells by reducing their metabolic rates.7-10 A reduction of hypothermia-induced cellular damage is necessary to extend the storage time of living cells for transplantation. Rubinsky et al. first demonstrated that fish AFPs protect rat liver,11 porcine oocyte,12 and bovine oocyte13 against hypothermic damage. Subsequently, Hays et al. reported that AFPs prevent the leakage of trapped solute as the artificial liposome passes through a phase transition.14 Tomczak et al. also showed that during chilling to nonfreezing temperatures, AFPs inhibit leakage from the liposome. They suggested that a hydrophobic interaction takes place between the AFPs and the lipid bilayer.15,16 However, it is not possible to apply the findings from the liposome studies to living cells because the cellular membrane is composed of various lipid and protein mixtures. Previously, we reported that the AFPs from notched-fin eelpout (NfeAFPs), Zoarces elongatus Kner, can protect cells during hypothermic storage.17 Although several applications of hypothermic preservations by AFPs have been reported,18,19 the detail of the preservation mechanism of living cells has not been clarified. The ability of AFPs was mainly measured after hypothermic preservation. There is little data available concerning the (8) Rauen, U.; de Groot, H. Cryobiology 2008, 56, 88–92. (9) Rauen, U.; Hintz, K.; Hanssen, M.; Lauchart, W.; Becker, H. D.; Degroot, H. Transplant Int. 1993, 6, 218–222. (10) Meng, Q. Biotechnol. Prog. 2003, 19, 1118–1127. (11) Lee, C. Y.; Rubinsky, B.; Fletcher, G. L. CryoLetters 1992, 13, 59–66. (12) Rubinsky, B.; Arav, A.; Mattioli, M.; Devries, A. L. Biochem. Biophys. Res. Commun. 1990, 173, 1369–1374. (13) Rubinsky, B.; Arav, A.; Fletcher, G. L. Biochem. Biophys. Res. Commun. 1991, 180, 566–571. (14) Hays, L. M.; Feeney, R. E.; Crowe, L. M.; Crowe, J. H.; Oliver, A. E. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 6835–6840. (15) Tomczak, M. M.; Hincha, D. K.; Crowe, J. H.; Harding, M. M.; Haymet, A. D. J. FEBS Lett. 2003, 551, 13–19. (16) Tomczak, M. M.; Hincha, D. K.; Estrada, S. D.; Wolkers, W. F.; Crowe, L. M.; Feeney, R. E.; Tablin, F.; Crowe, J. H. Biophys. J. 2002, 82, 874– 881. (17) Hirano, Y.; Nishimiya, Y.; Matsumoto, S.; Matsushita, M.; Todo, S.; Miura, A.; Komatsu, Y.; Tsuda, S. Cryobiology 2008, 57, 46–51. (18) Tablin, F.; Oliver, A. E.; Walker, N. J.; Crowe, L. M.; Crowe, J. H. J. Cell. Physiol. 1996, 168, 305–313. (19) Baguisi, A.; Arav, A.; Crosby, T. F.; Roche, J. F.; Boland, M. P. Theriogenology 1997, 48, 1017–1024.
Analytical Chemistry, Vol. 80, No. 23, December 1, 2008
9349
effects of AFPs on living cells during low-temperature incubation. Single cells are good experimental model systems for studying complex biological processes. However, obtaining information concerning their cellular state by optical microscopy is difficult. In general, fluorescence microscopy is used to observe cellular states, but monitoring of topographic change in cells is not easy due to photobleaching and photodamage.20,21 Atomic force microscopy (AFM) visualizes a cellular topography with high spatial resolution. The scanning probe frequently damages the target cell during continuous measurements because it must be positioned close to the cellular surface. On the other hand, scanning electrochemical microscopy (SECM) has the ability to evaluate single cells.22-24 SECM measurement permits the electrochemical profiling of the vicinity around active cells with submicrometer resolution. A microelectrode (∼25 µm) is used for measurement of the cytoplasmic membranes and evaluated topography,25,26 membrane permeability,27 photosynthetic activity,26 cell respiration,28,29 and metabolism30 of living cells. Since the current response of SECM shows a strong dependence on the probe-substrate distance, the regulation of the probe’s vertical position (z-position) is necessary. Some papers carried out SECM measurements with regulation of the probe’s z-position (constantdistance mode) by a tuning-fork,31-33 optical detection,34 and combined AFM.35 However, they have yet to accomplish longterm (from a few hours to a days), continuous monitoring of single cells. In the present study, we attempted to evaluate the preservation effects of AFPs on living cells during low temperature incubation. To assess the cellular state at constant low temperatures, we constructed an SECM system with temperature controls. In addition, we developed an automatic scanning procedure for the SECM system to carry out long-term, time-lapse imaging of single cells. EXPERIMENTAL SECTION Cell Lines and Materials. HepG2 (human hepatoma) cells were used in the present study. We grew the cells as a monolayer (20) Pepperkok, R.; Squire, A.; Geley, S.; Bastiaens, P. I. H. Curr. Biol. 1999, 9, 269–272. (21) Hopt, A.; Neher, E. Biophys. J. 2001, 80, 2029–2036. (22) Bard, A. J.; Mirkin, M. V. Scanning Electrochemical Microscopy; Marcel Dekker: New York, 2001. (23) Bard, A. J.; Li, X.; Zhan, W. Biosens. Bioelectron. 2006, 22, 461–472. (24) Schulte, A.; Schuhmann, W. Angew. Chem., Int. Ed. 2007, 46, 8760–8777. (25) Cai, C. X.; Liu, B.; Mirkin, M. V.; Frank, H. A.; Rusling, J. F. Anal. Chem. 2002, 74, 114–119. (26) Yasukawa, T.; Kaya, T.; Matsue, T. Anal. Chem. 1999, 71, 4637–4641. (27) Guo, J. D.; Amemiya, S. Anal. Chem. 2005, 77, 2147–2156. (28) Shiku, H.; Shiraishi, T.; Ohya, H.; Matsue, T.; Abe, H.; Hoshi, H.; Kobayashi, M. Anal. Chem. 2001, 73, 3751–3758. (29) Torisawa, Y. S.; Takagi, A.; Shiku, H.; Yasukawa, T.; Matsue, T. Oncol. Rep. 2005, 13, 1107–1112. (30) Liu, B.; Rotenberg, S. A.; Mirkin, M. V. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 9855–9860. (31) James, P. I.; Garfias-Mesias, L. F.; Moyer, P. J.; Smyrl, W. H. J. Electrochem. Soc. 1998, 145, L64–L66. (32) Oyamatsu, D.; Hirano, Y.; Kanaya, N.; Mase, Y.; Nishizawa, M.; Matsue, T. Bioelectrochemistry 2003, 60, 115–121. (33) Yamada, H.; Fukumoto, H.; Yokoyama, T.; Koike, T. Anal. Chem. 2005, 77, 1785–1790. (34) Hengstenberg, A.; Kranz, C.; Schuhmann, W. Chem.sEur. J. 2000, 6, 1547– 1554. (35) Macpherson, J. V.; Unwin, P. R. Anal. Chem. 2000, 72, 276–285.
9350
Analytical Chemistry, Vol. 80, No. 23, December 1, 2008
culture in flasks and subcultured them once a week at 37 °C in a humid atmosphere containing 5% CO2 in air, culturing them in high glucose (4.5 g/L) Dulbecco’s modified Eagle’s medium (Sigma, St. Louis, MO) supplemented with heat-inactivated 10% fetal bovine serum (Gibco, Carlsbad, CA). We obtained EuroCollins (EC) solution (15 mM KCl, 15 mM KH2PO4, 42 mM K2HPO4, 10 mM NaHCO3, and 194 mM glucose, pH 7.4) from I’ROM (Japan). EC solution is generally used for organ preservation as its intracellular ion composition and high concentration of glucose can be adjusted for osmotic pressure.36 Cultivated cells were grown in a 35 mm dish (Nippon Genetics, Japan) at a density of (1 × 104)-(3 × 104) cells/well and were cultured overnight. Before the experiment, we washed the cells with PBS (-) at room temperature and incubated them in preservation solution at 4 °C. All other reagents were analytical reagent grade, and solutions were prepared with ultrapure water obtained with a Milipore system. Antifreeze Proteins. NfeAFPs were purified from the muscles of notched-fin eelpout as described previously.37 Briefly, after removal of the head and gut, the flesh of the fish was homogenized with water in an electric mixer. We centrifuged the homogenate at 6000g for 30 min and dialyzed the obtained supernatant against 50 mM sodium citrate (pH 2.9) overnight at 4 °C. After removing the precipitate formed during dialysis, we loaded the AFP-containing solution into a High S column (1.0 cm × 5.0 cm; Bio-Rad, Hercules, CA) and eluted the column-bound AFPs with a linear NaCl gradient (0.0-0.5 M) in 50 mM sodium citrate (pH 2.9). The purified NfeAFPs were visualized as a single band after SDS-PAGE analysis and silver staining. The NfeAFPs consisted of a mixture of molecular weight isoforms (6.6-7.0 kDa). We dissolved lyophilized NfeAFPs sample in preservation solution to make 10 mg/mL concentrations in EC solution containing 4 mM K4[Fe(CN)6]. Instrumentation. We performed the SECM measurements on an instrument built in our laboratory using a piezo-motor positioning system and a potentiostat model HA1010 mM2B (Hokuto Denko, Japan). The SECM setup consisted of a PXI chassis with embedded controllers (PXI-1031 and PXI-8186 National Instruments (NI), Austin, TX) along with a data acquisition card (PXI-6289, NI). The data acquisition card was connected to the potentiostat and a temperature monitor by a thermocouple (Anritsu Keiki, Japan). A GPIB connector of PXI8186 connected to a XYZ stage controller (P-562.3CD, Physik Instrumente (PI), Germany). The XYZ stage can move 200 µm along the x-, y-, and z-axes with 10 nm resolution and is set on an inverted microscope (U-2000, Nikon, Japan). We constructed a temperature controlled bath on the microscope stage and connected it to the circulation unit with temperature control (CT-3000, EYELA, Japan). The software of the time-lapse SECM was constructed using LabView (NI). We used an Ag/AgCl (3 M KCl) electrode (Hokuto Denko, Japan) for the reference and auxiliary electrode. Electrochemical Measurements. We carried out all electrochemical measurements using EC solutions containing 4 mM K4[Fe(CN)6] with or without 10 mg/mL NfeAFPs. For long-term measurements, mineral oil (Sigma) was added to preservation (36) Muhlbacher, F.; Langer, F.; Mittermayer, C. Transplant. Proc. 1999, 31, 2069–2070. (37) Nishimiya, Y.; Sato, R.; Takamichi, M.; Miura, A.; Tsuda, S. FEBS J. 2005, 272, 482–492.
solutions to prevent evaporation because addition of mineral oil did not affect cellular activities (data not shown). We fabricated a Pt microdisk-electrode (5 µm radius) by heat sealing the Pt wire in a borosilicate glass capillary.38 The radius of the Pt electrode, including the glass sheath, was 10 µm. To determine the probe-substrate distance, we measured an approach curve on the substrate. The z-position of the microelectrode was determined from the approach curves based on the oxidation current of K4[Fe(CN)6] in the negative feedback mode. When the microelectrode is far from the substrate and a potential is applied, the steady state current (iT∞) is recorded. In the negative feedback results, a lower probe current (iT < iT∞) is observed when the tip is closer to the substrate (see Figure S-1 in the Supporting Information). Approach curves are presented in the dimensionless form of IT(L) versus (L), where IT(L) ) iT/ iT∞, L ) d/a, and dis the distance between the tip and the substrate and a is the electrode radius. We applied a potential of 0.5 V to the probe electrode for the oxidation of K4[Fe(CN)6] and moved the probe at a scan rate of 2 µm/s in order to determine the probe-substrate distance. We then evaluated the probe-substrate distance using the current profile and theoretical approach curve.39 Measurement Procedure of Time-Lapse SECM. Figure 1 shows a schematic procedure of the time-lapse SECM imaging. This procedure consists of four steps: namely, the determination of the probe-substrate distance (step 1), probe positioning (step 2), SECM imaging (step 3), and cellular height measurement (step 4). We positioned a Pt microelectrode on the substrate (dish surface) and then determined the z-position from the approach curves (step 1). When the oxidation current reached a specific value (It(L) ) 0.6-0.8, d ) 3-7 µm), the probe was stopped. After that, we withdrew the probe from the substrate and positioned it near the cell to be imaged (step 2). The probe-substrate distance was set to 19-21 µm. We performed the SECM measurements in an amperometric mode by keeping the potential at a constant value (step 3). When the SECM imaging was complete, the Pt microelectrode was withdrawn 30 µm from the imaging z-position. The SECM image provided the highest position of the cell whereby the probe could move over the center of the cell. After the probe moved, we measured an approach curve under the same conditions of step 1 (step 4). Since the lipid membrane of living cells is impermeable for a hydrophilic redox mediator,25,30 measurement currents mainly depend on a distance between the probe and the cellular surface. On the other hand, when the cellular membrane was injured by hypothermia, measurement currents depend on the probe-cell distance and cytoplasmic membrane permeability.40,41 The measurement current profiles of steps 1 and 4 provided a quantitative analysis of the observed cellular height. The distance between the measurement point on the dish (step 1) and the cell (step 4) was about 40 µm. The probe was moved to the initial position and maintained for a few moments until the start time of the next measurement. Prior to time-lapse imaging, we carried out a prescan (steps 1-4) of the target cell. After we confirmed that the dish and probe did not (38) Yasukawa, T.; Uchida, I.; Matsue, T. Biophys. J. 1999, 76, 1129–1135. (39) Shao, Y. H.; Mirkin, M. V. J. Phys. Chem. B 1998, 102, 9915–9921. (40) Barker, A. L.; Macpherson, J. V.; Slevin, C. J.; Unwin, P. R. J. Phys. Chem. B 1998, 102, 1586–1598. (41) Matsue, T.; Shiku, H.; Yamada, H.; Uchida, I. J. Phys. Chem. 1994, 98, 11001–11003.
Figure 1. Schematic procedure of time-lapse SECM for a single cell.
tilt from the prescan measurement, the time-lapse imaging was initiated. Initial cellular height was determined from the data obtained in the prescan measurement, and then the probesubstrate distance was determined. The following are the measurement condition of time-lapse SECM. We set the temperature to 4 °C and the probe potential to 0.5 V (versus Ag/AgCl). The scanning rates of the SECM imaging and the approach curve were 20 and 2 µm/s, respectively. The imaging area was 50 µm × 50 µm and XY resolution was 2 µm. The time between steps 1 and 4 was about 2 min 30 s. This measurement was automatically carried out every 3 min. RESULTS AND DISCUSSION Construction of the Time-Lapse SECM in Low Temperature. Figure 2 shows a schematic diagram of the temperature controlled time-lapse SECM. Because temperature control is absolutely required in this system, we introduced a temperature controlled bath with a thermocouple into the SECM system. After the HepG2 cells were incubated in preservation solutions at a Analytical Chemistry, Vol. 80, No. 23, December 1, 2008
9351
Figure 2. System setup of temperature controlled, time-lapse SECM. (A) Whole image of SECM system. (B) Schematic diagram on the microscope stage.
constant low temperature (4 °C), we selected and scanned a single target cell with the probe of SECM. However, the volume of the cell culture dish varied with a margin of error of ±1 °C when the temperature was controlled, so the distance between the probe and the substrate surface did not remain constant. Therefore, to achieve strict temperature control, we improved the circulation unit with temperature control to keep the incubation temperature within a margin of error of ±0.1 °C per h (see Figure S-2 in the Supporting Information). Considering another factor influencing the probe-substrate distance, there are various vibrations outside the measurement system. To correct the probe’s z-position, we automatically measured an approach curve in the negative feedback mode and adjusted the z-position of the microelectrode using the current profile of the approach curve before every imaging session (see Figure 1A,B). First, we carried out electrochemical measurements at various temperatures in EC solutions containing K4[Fe(CN)6] in the presence or absence of NfeAFPs and calculated the diffusion coefficients from the steady-state currents (see Figure 3). The oxidation current of K4[Fe(CN)6] depended on the temperature from 0 to 22 °C, and the presence of NfeAFPs had little influence on the electrochemical reaction. Even though the steadystate current at 4 °C decreased by half of the current at room temperature (22 °C), the current value was still sufficient to image cellular topography. Time-Lapse SECM Measurement of Cellular Topography during Low Temperature Incubation. HepG2 cells are cold sensitive, and previously, we reported that HepG2 became coldtolerant in preservation solutions containing NfeAFPs.17 We measured single cell images by time-lapse SECM to examine the cellular topography during cold incubation (4 °C). The addition of 4 mM K4[Fe(CN)6] did not inhibit the cellular preservation for 24 h (see Figure S-3 in the Supporting Information), and we incubated the cells in EC solutions with or without NfeAFPs and measured SECM images of HepG2 every 3 min for 24 h. Measurement currents were normalized by background current, which was obtained on the dish surface for every image. Figure 4A,B shows the time-lapse SECM images of the single cell, and 9352
Analytical Chemistry, Vol. 80, No. 23, December 1, 2008
Figure 3. Plot of the diffusion coefficient versus temperature in electrochemical measurements. The data was measured in EC solution containing 4 mM K4[Fe(CN)6] or EC solution containing 4 mM K4[Fe(CN)6] and 10 mg/mL NfeAFPs using the Pt microelectrode (φ ) 10 µm). We calculated the diffusion coefficient with the steadystate diffusion-controlled current.
Figure 4C shows the time course of the oxidation current, which is derived from the center of the cellular image. Initial-cellular height of HepG2 in the presence and the absence of NfeAFPs was about 8 and 9 µm, respectively. We set the probe-substrate distances to 19.7 and 20.7 µm from the substrate. When the cells were incubated only in EC solutions, the images did not change for 8 h, but the oxidation current on the cell rapidly decreased and reached a minimum value at 8.3 h (see Figure 4A,C). On the other hand, in the presence of NfeAFPs, there were no changes in the cell image for 16 h and then the current gradually decreased and reached a minimum value at 22.1 h (see Figure 4B,C). The current decrease indicated the increase in cellular volume because the currents on the cell depended on the distance between the probe and the cellular surface. The currents in both preservation solutions drastically increased after reaching the minimum value, and the increase corresponds to a decrease in cellular volume or an increase in membrane permeability. Previously, we reported that cold stress destroyed membrane integrity, resulting in release of intracellular lactate dehydrogenase within 24 h when using EC solution.17 Hence, we believe that the rapid increase in the current was induced by the rupture of the cytoplasmic membrane. Figure 4C shows the current values at the top of the cell at each incubation time. We calculated the cellular height more precisely using the approach curves during the cold incubation to accurately investigate cellular topographic changes. The difference of current profiles between those measured on the dish (see Figure 1A) and those on the center of the cell (see Figure 1 D) provided the observed cellular height (see Figure S-4 in the Supporting Information). Figure 5 shows the time courses of the cellular height of a single HepG2. When the cell was incubated in EC solution, the cellular height was constant over 8 h and then increased from 9 to 11 µm. In the presence of NfeAFPs, the cellular height did not change for 16 h and then gradually increased from 8 to 11 µm. After the maximum value was reached, the observed cellular height decreased again. As described above, the drastic reduction in observed cellular height was mainly caused by the increase in membrane permeability.
Figure 4. The time-lapse SECM images of a single HepG2 at low temperature. E ) 0.5 V vs Ag/AgCl; tip, Pt 10 µm microelectrode; scanning rate, 20 µm/s; imaging area, 50 µm; resolution, 2 µm. We measured these images in 4 mM K4[Fe(CN)6] and EC solution with or without 10 mg/mL NfeAFPs. The oxidation currents were normalized with the background current, which was obtained on the dish surface for every image. (A) The images calculated from the current value in the absence or presence of NfeAFPs. (B) The images depict from 18.1 to 22.6 h in the presence of NfeAFPs. (C) The time course of the oxidation current at the top of the cell. Inset: The gray arrows indicate the cellular swelling phase and the cell rupture points.
Figure 6. Schematic drawing of the cellular damaging process during cold incubation. Cells did not change size in the early phase (phase I), began to swell after a period of time (phase II), and finally ruptured.
Table 1. Analyses of Hypothermic Preserved Cells Using Time-Lapse SECM Figure 5. Time course of cellular height in low-temperature incubation. The cellular heights were calculated by the approach curves on the single HepG2 after SECM imaging.
These results demonstrate that the SECM measurements enabled dynamic imaging of living cellular topography for 24 h in 3 min intervals. The cellular volume could generally be evaluated using a Coulter counter, but time course measurement is difficult.42,43 Our system has several advantages compared to the other cellular status assay, including achieving direct observation of single cells and long-term continuous measurement. Preservation Effects of AFPs. The long-term measurement of single cells revealed that the cells change their size under hypothermic preservation. Next, we carried out long-term measurements of single cells in several independent experiments to investigate these cellular changes in more detail. In all measurements, we obtained the same dynamic images. Cells did not change their sizes in the early phase of preservation in either solution but did begin to swell after a period of time and finally ruptured. On the basis of these results, we classified the cellular changes in three phases during cold incubation, according (42) Conlon, I. J.; Dunn, G. A.; Mudge, A. W.; Raff, M. C. Nat. Cell Biol. 2001, 3, 918–921. (43) Rouzaire-Dubois, B.; Milandri, J. B.; Bostel, S.; Dubois, J. M. Pflugers Arch. 2000, 440, 881–888.
preservation solution ECa (n ) 8) NfeAFPsb/EC (n ) 5) c
phase I (h) phase (I + II) (h) 6.1 ± 4.0 19 ± 4.6c
7.0 ± 3.9 30 ± 9.2c
hmax/h0 1.22 ± 0.11 1.47 ± 0.24d
a Euro-Collins solution. b Antifreeze proteins from notched-fin eelpout. p < 0.001. d p < 0.05.
to the schematic drawing shown in Figure 6. We define phase I as the phase when the cellular height ratio (h/h0) is under 1.1, where h0 is the initial cellular height and h is the cellular height at the time of the measurement. The second phase (phase II) occurs between the end of phase I and cell rupture (the swelling phase). Table 1 summarizes the times of all phases and the h/h0. We performed a comparison between experimental groups using the Student’s t test. A p value of
