![DICR measurements in 96-well plates using a fluorescence microplate reader. (A) Schematic drawing of RyR1/R-CEPIA1er HEK293 cells without (No BV, left) and with (5 BV, right) BV infection of essential components for DICR (Cav1.1 carrying N617D Ca2+ non-conducting mutation, β1a, JP2, Stac3, and Kir2.1). (B) Typical results of time-lapse R-CEPIA1er fluorescence measurement using a FlexStation3 fluorescence microplate reader for RyR1/R-CEPIA1er HEK293 cells without (No BV, left) or with (5 BV, right) BV infection. High [K+] solution ranging from 5 to 61 mM (symbols shown in right) or 10 mM caffeine (light blue) was applied at 30 s after starting (blue arrowheads). (C) [K+] dependence of R-CEPIA1er fluorescence (F/F0) in No BV (black) and 5 BV cells (red). F/F0 was obtained by normalizing the fluorescence (F) at 35 s (open circles) or the averaged fluorescence of 125–150 s (filled circles) to that of the first 25 s (F0). Data are means ± SD (n = 9, N = 3 for 5 BV and n = 6, N = 3 for No BV). Note that [K+] dependent plot with data at 35 s (open red) was shifted rightward compared with that from data at 125–150 s (filled red) with larger variations, especially around EC50 values. (D) Fluorescence change by 61 mM [K+] or 10 mM caffeine. Fluorescence change is expressed as 1 − F/F0 (see C). Data are means ± SD (n = 6, N = 3) and were analyzed by two-way ANOVA with Bonferroni’s multiple comparisons test. Note that 5 BV cells show large fluorescence change with high [K+] which is nearly 80% of that with caffeine. n is the number of wells and N is the number of independent experiments.](https://cdn.rupress.org/rup/content_public/journal/jgp/154/12/10.1085_jgp.202213230/2/jgp_202213230_fig1.png?Expires=1773695562&Signature=ahdLsaSYVdk~hZt8Ogwo0hGTp1~rn1OMEzb6CDR1SvZLJtPLqbHXOgHl1CmfebJE0iseUvYPpNwIaQLd7y6Miiq0-LVRU4lu1FhTTufE9XW-Qhn1NSSZx3otEoC9aMk3nQlqdbj~KeFjb3XoptLnFyaZ6iSWofgux1CAI770kY8XM9mZItlmP8rWowujnluH66zyhrORG4f5LsgqL6RwjGEF~KrxevkNlyPLaJR1TcOvTeCafFxXBbMq7S9UP~rxFCEBJh0KfKEFprnv1laQdYIfSCsBYN6ysPbEQXGLquH2hWDWYiVVrYj0Dbu0S-v4BV0wMzPvaqi83nT0lKaWNg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
DICR measurements in 96-well plates using a fluorescence microplate reader. (A) Schematic drawing of RyR1/R-CEPIA1er HEK293 cells without (No BV, left) and with (5 BV, right) BV infection of essential components for DICR (Cav1.1 carrying N617D Ca2+ non-conducting mutation, β1a, JP2, Stac3, and Kir2.1). (B) Typical results of time-lapse R-CEPIA1er fluorescence measurement using a FlexStation3 fluorescence microplate reader for RyR1/R-CEPIA1er HEK293 cells without (No BV, left) or with (5 BV, right) BV infection. High [K+] solution ranging from 5 to 61 mM (symbols shown in right) or 10 mM caffeine (light blue) was applied at 30 s after starting (blue arrowheads). (C) [K+] dependence of R-CEPIA1er fluorescence (F/F0) in No BV (black) and 5 BV cells (red). F/F0 was obtained by normalizing the fluorescence (F) at 35 s (open circles) or the averaged fluorescence of 125–150 s (filled circles) to that of the first 25 s (F0). Data are means ± SD (n = 9, N = 3 for 5 BV and n = 6, N = 3 for No BV). Note that [K+] dependent plot with data at 35 s (open red) was shifted rightward compared with that from data at 125–150 s (filled red) with larger variations, especially around EC50 values. (D) Fluorescence change by 61 mM [K+] or 10 mM caffeine. Fluorescence change is expressed as 1 − F/F0 (see C). Data are means ± SD (n = 6, N = 3) and were analyzed by two-way ANOVA with Bonferroni’s multiple comparisons test. Note that 5 BV cells show large fluorescence change with high [K+] which is nearly 80% of that with caffeine. n is the number of wells and N is the number of independent experiments.
