TY - JOUR
T1 - A flow cytometric method for measurement of intracellular chloride concentration in lymphocytes using the halide-specific probe 6-methoxy-N-(3- sulfopropyl) quinolinium (SPQ)
AU - Pilas, Barbara
AU - Durack, Gary
PY - 1997
Y1 - 1997
N2 - A flow cytometry method using the halide-specific fluorescent dye, 6- methoxy-N-(3-sulfopropyl) quinolinium (SPQ), has been developed to measure intracellular chloride concentration in single cells. Collisions with chloride quench the fluorescence of SPQ, making it possible to relate the measured fluorescence intensity to chloride concentration with a Stern- Volmer equation. To demonstrate the method, porcine lymphocytes were loaded in vitro, using a hypotonic method, with 5 mM SPQ. Fluorescence excitation was provided by a UV laser and the fluorescence emission intensity at 485 nm was recorded. Calibration was performed by using 7 μM nigericin (a K/H antiporter) and 10 μM tributyltin (a Cl/OH antiporter) to equilibrate the concentrations of intracellular and extracellular chloride. Calibration measurements were made for chloride concentrations between 0 mM and 140 mM. The calibration produced a Stern-Volmer quenching constant of 16.2 M-1 which was used to relate measured cell fluorescence to intracellular chloride concentration. The intracellular chloride concentration for fresh porcine lymphocytes was determined to be 56.2 ± 3.3 mM. Stable loading of cells with 5 mM SPQ was accomplished in 15 minutes, leakage of SPQ from the cells was minimal, and over 95% of the cells remained viable after loading.
AB - A flow cytometry method using the halide-specific fluorescent dye, 6- methoxy-N-(3-sulfopropyl) quinolinium (SPQ), has been developed to measure intracellular chloride concentration in single cells. Collisions with chloride quench the fluorescence of SPQ, making it possible to relate the measured fluorescence intensity to chloride concentration with a Stern- Volmer equation. To demonstrate the method, porcine lymphocytes were loaded in vitro, using a hypotonic method, with 5 mM SPQ. Fluorescence excitation was provided by a UV laser and the fluorescence emission intensity at 485 nm was recorded. Calibration was performed by using 7 μM nigericin (a K/H antiporter) and 10 μM tributyltin (a Cl/OH antiporter) to equilibrate the concentrations of intracellular and extracellular chloride. Calibration measurements were made for chloride concentrations between 0 mM and 140 mM. The calibration produced a Stern-Volmer quenching constant of 16.2 M-1 which was used to relate measured cell fluorescence to intracellular chloride concentration. The intracellular chloride concentration for fresh porcine lymphocytes was determined to be 56.2 ± 3.3 mM. Stable loading of cells with 5 mM SPQ was accomplished in 15 minutes, leakage of SPQ from the cells was minimal, and over 95% of the cells remained viable after loading.
KW - Chloride concentration
KW - Fluorescence quenching
KW - SPQ
UR - http://www.scopus.com/inward/record.url?scp=0030798074&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0030798074&partnerID=8YFLogxK
U2 - 10.1002/(SICI)1097-0320(19970801)28:4<316::AID-CYTO7>3.0.CO;2-9
DO - 10.1002/(SICI)1097-0320(19970801)28:4<316::AID-CYTO7>3.0.CO;2-9
M3 - Article
C2 - 9266752
AN - SCOPUS:0030798074
SN - 0196-4763
VL - 28
SP - 316
EP - 322
JO - Cytometry
JF - Cytometry
IS - 4
ER -