(A) HeLa cells, (C) a HEK-293 cell and (E) a U2-OS cell co-expressing ER-LAR-GECO1 (red) and CatchER (green). Scale bar=20 μm. (B, D and F) Effects of depleting the ER of Ca²⁺ by addition of thapsigargin (10 μM) on the fluorescence intensities of ER-LAR-GECO1 (red line) and CatchER (green line) co-expressed in a HeLa cell, a HEK-293 cell and a U2-OS cell as shown in (A), (C) and (E), respectively. Traces show results from single cells, typical of at least four similar recordings. (G) A HeLa cell co-expressing ER-LAR-GECO1 (red) and ER-GAP1 (blue, 395 nm excitation; green, 488 nm excitation). Scale bar=20 μm. (H) Effects of the release of ER Ca²⁺ evoked by histamine (100 μM) and ionomycin (Iono, 10 μM) on the fluorescence intensity of ER-LAR-GECO1 (F/F₀) and the ratiometric fluorescence response of ER-GAP1 (R/R₀) co-expressed in a HeLa cell. Traces are from a single cell, typical of at least four similar recordings.