Cells are seeded 24 h prior to experiments on 10 x 50-mm, HCl- and ethanol-washed coverslips to a confluency of 70 to 90% at the time of the experiments. Cells are loaded with Fura-2 by preincubation with 2 mM Fura-2 acetoxymethyl ester (Fura-2-AM) in standard isotonic medium for 20 min at 37° followed by a wash and a 15-min postloading incubation to ensure proper intracellular cleavage of the acetoxymethylester groups to obtain Fura-2.
We employ a PTI RatioMaster spectrophotometer equipped with a standard photomultiplier (PMT) system, excitation and emission monochrometers for wavelength selection, a 75-W xenon lamp, and a temperature-controlled cuvette house. During experiments, the cuvette is perfused continuously with preheated (37°) standard Ringer's solutions at a speed of about 0.7 ml/min, increasing to 3.5ml/min during solution changes for rapid complete exchange in the cuvette. The coverslip with the cells is placed at a 50° angle relative to the excitation light, and scattered light is collected at an angle of90° relative to the excitation light. Excitation and emission wavelengths for light scattering need to be optimized for each cell type used. Generally, the best light-scattering signal is obtained after excitation in the wavelength range of570 to 600 nm. The emission wavelength is set about 5 nm red shifted to the excitation wavelength to protect the PMT from excitation light. To the extent that the cells swell as near-perfect osmometers, and in the absence of volume regulation, the light-scattering signal will be a linear function ofthe osmolarity. Practically, this is achieved by very rapid solution changes, such that volume regulation is negligible in the time window studied.
Figure 10.3A and B show an experiment evaluating the linearity of the light-scattering response to osmolarity changes in Ehrlich-Lettre ascites (ELA) murine tumor cells. With these cells, the optimal light-scattering signal in our setup is obtained at 589 nm excitation, and emission is measured at 595 nm. Figure 10.3A is a representative trace, and it is seen that the light-scattering signal is directly related to extracellular osmolarity, that is, inversely related to cell volume. Therefore, data are calculated as the inverse of the light-scattering signal relative to that obtained in the initial, isotonic condition (I0), that is, 1/(I/Iq), or I0/I. Figure 10.3B shows Iq/I from the experiment in Fig. 10.3A as a function of extracellular osmolality. Figures 10.3C and 10.3D show the light-scattering signal, and Iq/I, for an
Figure 10.3 The use of large-angle light scattering to monitor cell volume changes. (A) Light scattering as a function of extracellular osmolarity in Ehrlich Lettre ascites cells. Cells were seeded 24 h prior to experiments on 10 X 50-mm coverslips to a confluency of about 70% at the time of the experiments. Cells were mounted in a temperature-controlled cuvette in a PTI RatioMaster spectrophotometer and were perfused continuously with preheated (37°) Ringer's solutions, which were changed rapidly to the osmolarity indicated by increasing the perfusion rate from 0.7 to 3.5 ml/min. Excitation was measured at 589 nm excitation, and emission is measured at 595 nm. (B) Data from A were converted to relative cell volumes by calculating the inverse of the light-scattering signal relative to that obtained in the initial, isotonic condition (Iq), i.e.,1/(I/I0), or Iq/I. (C andD) Raw data and Iq/I for an experiment assessing RVD in about 90% confluent ELA cells after a 35% reduction in extracellular osmolarity. The experiment was carried out as described in A. It may be noted that the magnitude ofthe light-scattering signal, as well as of the relative changes in light scattering, is strongly cell density dependent, hence only populations of equal confluency should be compared.
experiment assessing RVD in ELA cells after a 35% reduction in extracellular osmolarity. RVD may be calculated as the slope ofthe initial, linear part of the relative cell volume traces following maximal cell swelling (for further examples, see Pedersen et al., 2002).
The simultaneous assessments of Fura-2 fluorescence are carried out by measuring at 510 nm after excitation at 340 and 380 nm. Practically, this is achieved by running continuous cycles of excitation and measurement, such that cells are excited at 589 nm and emission is measured at 595 nm, followed by excitation at 340 and 380 nm, respectively, and emission measurement at 510 nm. Fura-2 data are evaluated as the 340/380-nm ratio after background subtraction and may be converted to [Ca2+]i values by in vitro calibration as described previously (Grynkiewicz et al., 1985). In our hands, Fura-2 loading has no effect on the light-scattering measurements.
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