The fluorescent dye MQAE was used to determine [Cl
-]
i as described by Rocha-Gonzalez [
40] with some modifications [
8]. Cells were incubated with 5 mM MQAE for 1–2 h (37°C) in a HEPES buffered isotonic solution. The HEPES buffered isotonic solution contained (in mM, pH 7.4): 100 NaCl, 5.4 KCl, 1.3 CaCl
2, 0.8 MgSO
4, 20 HEPES, 5.5 glucose, 0.4 NaHC0
3, and 70 sucrose with 310 mOsm determined with an osmometer (Advanced Instruments, Norwood, MA). The coverslip was placed in the heated imaging chamber for 30 min before imaging. Using the Nikon TiE inverted epifluorescence microscope and the 40× oil immersion objective lens, cells were excited every 60 sec at 340 and emission fluorescence at 460 nm recorded. Images were collected and analyzed with the MetaFluor image-processing software. At the end of each experiment, the MQAE florescence was calibrated under a steady state condition when [Cl
-]
o and [Cl
-]
i were considered equal by exposing cells to a series of calibration solutions containing 10 μM tributylin and 5 μM nigericin (Rocha-Gonzalez, Mao, and Alvarez-Leefmans 169–84). The series of Cl
- calibration solutions contained (in mM): 1.27 Ca(OH)
2, 0.8 MgSO
4, 5 HEPES, 5.5 glucose, 120 K
+, and variable Cl
- and NO
3-. In these solutions, Cl
- was varied from 0 to 60 mM keeping the sum of Cl
- and NO
3- equal to 120 mM. KSCN (150 mM) was used to quench the MQAE fluorescence, which was taken as background fluorescence. [Cl
-]
i was determined from the MQAE fluorescence (drift-corrected, background-corrected) using the following equation: [
Cl-]
i
= [(
F
o
/
F
t
) - 1)]/
Ksv, where F
o was the fluorescence in 0 mM [Cl
-]
o, Ft was the fluorescence at any given time point, and K
sv was the slope of the linear fit of MQAE fluorescence vs. the [Cl
-]
o of the standards. A K
sv of 13.4 ± 1.5 M
-1 was calculated in our study [
8], a value similar to that reported by others [
40].