Figure 1
Figure 1. Hypotonic shock-induced ATP release tightly correlates with cell lysis. (A) ATP content in RBC supernatants exposed for 5 minutes to isotonic (control) or 25%, 30%, or 37% hypotonic solutions in 6-well plate format. The data are average ± SEM (n = 3 determinations), and, for hypotonic conditions, they are significantly different from control values (P < .05, Mann-Whitney U test, indicated by *). (B) Absorbance spectra (λ = 360-700 nm) of RBC supernatants exposed to different hypotonic solutions from the experiment reported in A. Absorbance of supernatant from cell lysates equivalent to 100, 1000, and 5000 cells/µL is also shown. Note that the absorbance spectra of 30% hypotonic stress supernatants overlap with those of lysates of 1000 cells/µL. From the calibration curve of peak absorbance (at 414 nm) vs cell lysate density, as illustrated in supplemental Figure 1B, we could calculate the number and percentage of lysed cells in each experimental condition. (C) For low hemolysis levels, lysed cell number was evaluated more precisely by plotting the entire absorbance spectra of the supernatant vs the absorbance spectra of the reference lysate. y-axis: Absorbance spectra of RBC supernatants exposed to different hypotonic solutions from the experiments reported in A are plotted against absorbance of reference lysate of 1000 cells/µL (x-axis) for the entire spectrum between 380 and 620 nm. From the slope of linear fit, the exact number of lysed cells in the supernatants was determined for each experimental condition: Iso, Hypo 25%, 30%, and 37%. The corresponding number of lysed cells was 226, 603, 1073, and 6470/µL, respectively. (D) Hypotonic shock-induced ATP release vs number of lysed cells. The number of lysed cells in control (Iso) and hypotonic conditions was calculated by fitting their corresponding absorbance spectra to those of a reference cell lysate of 1000 cells/µL (white circles), shown in C. The solid line is a least square linear fit to the hypotonic shock-induced ATP release data with 95% confidence bands indicated by dashed lines and correlation coefficient R = 0.988. Red triangles indicate ATP measured in cell lysates prepared from the same batch of blood and adjusted to corresponding numbers of lysed cells found under Iso and hypotonic conditions. The slope of the linear relationship corresponds to an intracellular ATP concentration of 1.42 mM. (E) Correlation between extent of hemolysis (shown as absorbance – left axis, or % hemolysis – right axis) and ATP release induced by hypotonic shock. Paired values of extracellular ATP and free hemoglobin in the supernatant samples were fitted by linear regression with Origin Laboratory 7.5. The data are examples of 3 independent experiments (each indicated by a different symbol) similar to those in A-D but performed in 12-well plate format. In each experiment, RBC suspensions were incubated in either isotonic or hypotonic (20%, 25%, 30%, 35%, or 40%) solution. The slopes of the fitted lines correspond to the intracellular ATP concentration and are indicated on the graph. (F) Effect of CBX (100 μM) on ATP release stimulated by 5-minute exposure to 25% hypotonic shock. Cells were preincubated for 10 minutes with CBX in isotonic PSS, then transferred to a Petri dish containing CBX in hypotonic medium. Average of n = 3 experiments ± SEM.

Hypotonic shock-induced ATP release tightly correlates with cell lysis. (A) ATP content in RBC supernatants exposed for 5 minutes to isotonic (control) or 25%, 30%, or 37% hypotonic solutions in 6-well plate format. The data are average ± SEM (n = 3 determinations), and, for hypotonic conditions, they are significantly different from control values (P < .05, Mann-Whitney U test, indicated by *). (B) Absorbance spectra (λ = 360-700 nm) of RBC supernatants exposed to different hypotonic solutions from the experiment reported in A. Absorbance of supernatant from cell lysates equivalent to 100, 1000, and 5000 cells/µL is also shown. Note that the absorbance spectra of 30% hypotonic stress supernatants overlap with those of lysates of 1000 cells/µL. From the calibration curve of peak absorbance (at 414 nm) vs cell lysate density, as illustrated in supplemental Figure 1B, we could calculate the number and percentage of lysed cells in each experimental condition. (C) For low hemolysis levels, lysed cell number was evaluated more precisely by plotting the entire absorbance spectra of the supernatant vs the absorbance spectra of the reference lysate. y-axis: Absorbance spectra of RBC supernatants exposed to different hypotonic solutions from the experiments reported in A are plotted against absorbance of reference lysate of 1000 cells/µL (x-axis) for the entire spectrum between 380 and 620 nm. From the slope of linear fit, the exact number of lysed cells in the supernatants was determined for each experimental condition: Iso, Hypo 25%, 30%, and 37%. The corresponding number of lysed cells was 226, 603, 1073, and 6470/µL, respectively. (D) Hypotonic shock-induced ATP release vs number of lysed cells. The number of lysed cells in control (Iso) and hypotonic conditions was calculated by fitting their corresponding absorbance spectra to those of a reference cell lysate of 1000 cells/µL (white circles), shown in C. The solid line is a least square linear fit to the hypotonic shock-induced ATP release data with 95% confidence bands indicated by dashed lines and correlation coefficient R = 0.988. Red triangles indicate ATP measured in cell lysates prepared from the same batch of blood and adjusted to corresponding numbers of lysed cells found under Iso and hypotonic conditions. The slope of the linear relationship corresponds to an intracellular ATP concentration of 1.42 mM. (E) Correlation between extent of hemolysis (shown as absorbance – left axis, or % hemolysis – right axis) and ATP release induced by hypotonic shock. Paired values of extracellular ATP and free hemoglobin in the supernatant samples were fitted by linear regression with Origin Laboratory 7.5. The data are examples of 3 independent experiments (each indicated by a different symbol) similar to those in A-D but performed in 12-well plate format. In each experiment, RBC suspensions were incubated in either isotonic or hypotonic (20%, 25%, 30%, 35%, or 40%) solution. The slopes of the fitted lines correspond to the intracellular ATP concentration and are indicated on the graph. (F) Effect of CBX (100 μM) on ATP release stimulated by 5-minute exposure to 25% hypotonic shock. Cells were preincubated for 10 minutes with CBX in isotonic PSS, then transferred to a Petri dish containing CBX in hypotonic medium. Average of n = 3 experiments ± SEM.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal