TY - JOUR
T1 - Simultaneous analysis of steady-state intracellular pH and cell morphology by automated laser scanning cytometry
AU - Koo, Mei K.
AU - Oh, Chia H.
AU - Holme, Andrea L.
AU - Pervaiz, Shazib
N1 - Funding Information:
Biomedical Research Council (BMRC)
National Medical Research Council (NMRC) of Singapore
Acknowledgements:
We thank Kok Tee Toh for help with flow cytometer.
PY - 2007/2/1
Y1 - 2007/2/1
N2 - Background: Cytosolic pH (pHi) changes are critical in cellular response to diverse stimuli, including cell survival and death signaling. The potential drawback in flow-based analysis is the inability to simultaneously visualize the cells during pHi measurements. Here, the suitability of laser scanning cytometer (LSC) in pHi measurement was investigated. Aim: Using the two extensively reported pH-sensitive fluorescent probes, 2,7-bis(2-Carboxyethyl)- 5(6)carboxyfluorescein acetoxymethyl ester (BCECF-AM) and 5-(and-6)carboxy SNARF-1 acetoxymethyl ester, we evaluated the potential of automated LSC as a platform for simultaneous determination of pHi and cell morphology. The effect of a variety of buffer systems-commonly employed for pHi measurements-on cell morphology before pH clamping with the ionophore, nigericin, was also assessed. Methods: Measurement of cytosolic pH was performed using pH-sensitive fluorescent probes BCECF-AM and SNARF-1. pH clamping was carried out using nigericin and samples were analyzed on the LSC or CyAn™ ADP Flow Cytometer. Results: The pHi clamping conditions were optimized as 140 mM potassium and 10 μM nigericin. The suitable buffers used for pH clamping: 140 mM KCl, 1 mM MgCl2, 2 mM CaCl2·2H2O, 5 mM glucose, 20 mM MES and 140 mM KCl, 1 mM MgCl2, 2 mM CaCl 2·2H2O, 5 mM glucose, and 20 mM Tris. Results obtained with the LSC strongly correlated with those obtained by flow cytometry. Conclusion: We report here that LSC is an excellent and highly reproducible platform for pHi determination, and provides the added advantage of simultaneous imaging of cells before, during, and after pH measurements.
AB - Background: Cytosolic pH (pHi) changes are critical in cellular response to diverse stimuli, including cell survival and death signaling. The potential drawback in flow-based analysis is the inability to simultaneously visualize the cells during pHi measurements. Here, the suitability of laser scanning cytometer (LSC) in pHi measurement was investigated. Aim: Using the two extensively reported pH-sensitive fluorescent probes, 2,7-bis(2-Carboxyethyl)- 5(6)carboxyfluorescein acetoxymethyl ester (BCECF-AM) and 5-(and-6)carboxy SNARF-1 acetoxymethyl ester, we evaluated the potential of automated LSC as a platform for simultaneous determination of pHi and cell morphology. The effect of a variety of buffer systems-commonly employed for pHi measurements-on cell morphology before pH clamping with the ionophore, nigericin, was also assessed. Methods: Measurement of cytosolic pH was performed using pH-sensitive fluorescent probes BCECF-AM and SNARF-1. pH clamping was carried out using nigericin and samples were analyzed on the LSC or CyAn™ ADP Flow Cytometer. Results: The pHi clamping conditions were optimized as 140 mM potassium and 10 μM nigericin. The suitable buffers used for pH clamping: 140 mM KCl, 1 mM MgCl2, 2 mM CaCl2·2H2O, 5 mM glucose, 20 mM MES and 140 mM KCl, 1 mM MgCl2, 2 mM CaCl 2·2H2O, 5 mM glucose, and 20 mM Tris. Results obtained with the LSC strongly correlated with those obtained by flow cytometry. Conclusion: We report here that LSC is an excellent and highly reproducible platform for pHi determination, and provides the added advantage of simultaneous imaging of cells before, during, and after pH measurements.
KW - BCECF-AM
KW - Intracellular pH
KW - Laser scanning cytometry
KW - Morphology
KW - SNARF-1
UR - http://www.scopus.com/inward/record.url?scp=33846922314&partnerID=8YFLogxK
U2 - 10.1002/cyto.a.20361
DO - 10.1002/cyto.a.20361
M3 - Article
C2 - 17200954
AN - SCOPUS:33846922314
VL - 71
SP - 87
EP - 93
JO - Cytometry Part A : Journal of the International Society for Analytical Cytology
JF - Cytometry Part A : Journal of the International Society for Analytical Cytology
SN - 1552-4922
IS - 2
ER -