The pHi of ArchT transfected
RPE cells and control RPE cells were directly calculated using 2′,7′-bis(carboxyethyl)-5(6)-Carboxyfluorescein
(BCECF, Biotium). BCECF is a ratiometric dual excitation/single-emission
pH biosensor (405 ex/525 em for pH insensitive fluorescence, 488 ex/525
em for pH-sensitive fluorescence). Cells were loaded with 1 μM
BCECF for 10 min, washed with complete media 3 × 5 min. We performed
ratiometric imaging of cells loaded with BCECF dye (10 min, 37 C,
5% CO2). For standardization, pH standard buffers (pH ∼6.5
and ∼7.5 (0.025 M HEPES, 0.105 M KCl, 0.001 MgCl2)) were prepared with 10 μM nigericin (Invitrogen, N1495) (a
protonophore) and added sequentially to cells to equilibrate pHe and
pHi as previously described.28 (link) Individual
cell pHi was back-calculated using single-cell standard curves generated
from ratiometric fluorescence in nigericin standard buffers and pHi
values were reported inFigure 1 B.
RPE cells and control RPE cells were directly calculated using 2′,7′-bis(carboxyethyl)-5(6)-Carboxyfluorescein
(BCECF, Biotium). BCECF is a ratiometric dual excitation/single-emission
pH biosensor (405 ex/525 em for pH insensitive fluorescence, 488 ex/525
em for pH-sensitive fluorescence). Cells were loaded with 1 μM
BCECF for 10 min, washed with complete media 3 × 5 min. We performed
ratiometric imaging of cells loaded with BCECF dye (10 min, 37 C,
5% CO2). For standardization, pH standard buffers (pH ∼6.5
and ∼7.5 (0.025 M HEPES, 0.105 M KCl, 0.001 MgCl2)) were prepared with 10 μM nigericin (Invitrogen, N1495) (a
protonophore) and added sequentially to cells to equilibrate pHe and
pHi as previously described.28 (link) Individual
cell pHi was back-calculated using single-cell standard curves generated
from ratiometric fluorescence in nigericin standard buffers and pHi
values were reported in
