Calcium titration of G-GECO1.2 was performed by Calcium Calibration Buffer Kit #1 (Invitrogen). For calcium titration of low affinity mutants, a series of zero to 10 mM [Ca2+]free buffer were made in 1 mM EGTA, 50 mM MOPS, and 100 mM KCl (pH 7.2) and [Ca2+]free concentrations were calculated using WEBMAXC EXTENDED program (maxchelator.stanford.edu). The fluorescence of 1 µM purified protein in various [Ca2+]free buffers were measured with excitation at 485/20 nm and emission at 516/20 nm using a Synergy 2 Microplate Reader (Biotek).
Calcium calibration buffer kit 1
Manufactured by Thermo Fisher Scientific
The Calcium Calibration Buffer Kit #1 is a set of pre-mixed solutions designed for the calibration of instruments used to measure calcium levels. The kit contains a series of aqueous buffer solutions with known calcium concentrations, allowing users to verify and adjust the calibration of their measurement devices.
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Lab products found in correlation
B per, by Thermo Fisher Scientific (2 mentions)
T7 express e coli cells, by New England Biolabs (2 mentions)
Profinity imac resin, by Bio-Rad (2 mentions)
Safire2 plate reader, by Tecan (2 mentions)
Synergy 2 microplate reader, by Agilent Technologies (1 mentions)
Alexa 594, by Thermo Fisher Scientific (1 mentions)
Oregon green bapta 1, by Thermo Fisher Scientific (1 mentions)
Ogb 1, by Thermo Fisher Scientific (1 mentions)
Ff02 520 28, by IDEX Corporation (1 mentions)
Fusion α, by PerkinElmer (1 mentions)
96 well plate, by PerkinElmer (1 mentions)
Originpro 7, by OriginLab (1 mentions)
Prism 8, by GraphPad (1 mentions)
Standard treated glass capillaries, by NanoTemper (1 mentions)
Monolith nt 115, by NanoTemper (1 mentions)
Histamine, by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
9 protocols using calcium calibration buffer kit 1
1
Calcium Titration of G-GECO1.2 Sensor
2
Cytosolic Ca2+ Measurements in DA Neurons
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For cytosolic Ca2+ measurements in tissue slices, DA neurons were loaded with Alexa-594 (red dye, 30 µM, Invitrogen) and Oregon Green Bapta-1 (green dye, OGB-1, 200 µM, Invitrogen) together. Optical signals were acquired at 800 nm of two-photon excitation beam to simultaneously excite both dyes. ROI images were acquired during frame scanning (512 × 512 pixels) with 10–20 ms time intervals and Ca2+ levels from the ROIs were quantified as changes in green Ca2+ fluorescence from OGB-1 divided by morphological red fluorescence of Alexa-594 (G/R). Ratio between OGB-1 and Alexa-594 was used to minimize interference of the fluorescence photobleaching.
Acutely dissociated DA neurons were incubated with 3–5 µM Fluo 4-AM in high-glucose solution at room temperature (20°C–25°C) for 30 min. The fluorescence intensities of the neurons were measured using a Zeiss 510 confocal microscope (40× oil immersion objective lens or 60× water immersion objective lens). Fluo 4-AM Ca2+ indicators were excited at 488 nm (argon laser) and cytosolic Ca2+ signals were collected through 550 nm long-pass filter. Ca2+ level changes represented delta fluorescence intensity devided by basal level of fluorescence (ΔF/F0). To measure cytosolic Ca2+ concentration in some cases, we used a calibration kit (Calcium Calibration Buffer Kit #1; Invitrogen).
Acutely dissociated DA neurons were incubated with 3–5 µM Fluo 4-AM in high-glucose solution at room temperature (20°C–25°C) for 30 min. The fluorescence intensities of the neurons were measured using a Zeiss 510 confocal microscope (40× oil immersion objective lens or 60× water immersion objective lens). Fluo 4-AM Ca2+ indicators were excited at 488 nm (argon laser) and cytosolic Ca2+ signals were collected through 550 nm long-pass filter. Ca2+ level changes represented delta fluorescence intensity devided by basal level of fluorescence (ΔF/F0). To measure cytosolic Ca2+ concentration in some cases, we used a calibration kit (Calcium Calibration Buffer Kit #1; Invitrogen).
3
Calcium-Dependent Protein Folding Analysis
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Purified Tq-Ca-FLITS was 4× diluted in calcium buffers containing 0 or 39 µM free calcium of the Calcium Calibration Buffer Kit #1 (C3008MP, Thermo Fisher Scientific). The absorbance spectra were measured before and >5 min after addition of 1 M NaOH, at 260–650 nm with 1 nm step size and 1 nm bandwidth. Corresponding buffer was used as reference. The concentration of unfolded protein was determined using the Beer–Lambert law and assuming an extinction coefficient (ε) at 462 nm of 46 mM−1 cm−1 for the free cyan chromophore63 (link). Next, ε was determined at 440 nm for the calcium-free and -bound states. The average absorbance spectra of three measurements were plotted.
4
Fluorometric calcium indicator calibration
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In vitro fluorometry was performed in 96-well plates (PerkinElmer) using a Fusion α plate reader (PerkinElmer) at room temperature. Filter sets used for fluorescent measurement for green calcium indicators were ET495/10x (Chroma) (excitation) and FF02-520/28 (Semrock) (emission). The dynamic range was calculated as ΔF/F. ΔF/F was calculated as (Fmax–F0)/F0, where Fmax is the fluorescence intensity at a saturating [Ca2+] of 0.3 mM and F0 is the fluorescence intensity measured at zero [Ca2+] in the presence of 15 mM EGTA. Calcium titration experiments were performed by mixing 10 mM solutions of K2H2EGTA and Ca2EGTA proportionally from Calcium Calibration Buffer Kit #1 (Thermo Fisher Scientific). The Kd value and Hill coefficient were calculated by fitting according to the Hill equation using Origin Pro 7.5 (Origin Lab).
5
Characterizing Calcium Sensor Dynamics
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After whole-cell recording of an HEK 293A cell cotransfected with Orai1-GCaMP6f and mCherry-STIM1, the recording pipette was withdrawn and the tip was broken on the coverslip, forming a sharp edge. The cell was unroofed by carefully puncturing and scraping the broken pipette across the top surface of the cell under transmitted light illumination. Standard buffered Ca2+ solutions (Calcium Calibration Buffer Kit #1; Thermo Fisher Scientific) with K+ as the primary cation and varying free Ca2+ concentrations were used in probe calibration runs. The kit consisted of two solutions containing 10 mM K2EGTA (solution 1) and 10 mM CaEGTA (solution 2), and 100 mM KCl and 30 mM MOPS, pH 7.2 (both solutions). Mixing of these solutions at different ratios generated standard Ca2+ buffers ranging from 0 to 39 µM free Ca2+. These standard solutions were applied in series to the unroofed cell through a gravity-driven local perfusion system while two-channel fluorescence images were acquired. Unroofed cells were equilibrated in the perfusion stream for 20–30 s before image acquisition. At each Ca2+ concentration, green channel measurements were produced by subtracting the 0 nM Ca2+ measurement. Cell footprint ROIs were traced manually, and mean fluorescence values were measured. Fluorescence values were plotted, and Kd and Hill coefficients (hc) were calculated using Prism 8 (GraphPad Software).
6
Fluorescent Labeling of MtSEO-F1 Protein
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Purified MtSEO‐F1 protein was labeled with the red fluorescent dye NT‐647‐NHS using the MO‐L001 Monolith Protein Labeling Kit RED‐NHS (NanoTemper Technologies, Munich, Germany). The labeled protein solution was supplemented with 0.05% Tween‐20 and diluted to a concentration of 182.5 nM by mixing with an equal volume of buffers containing different concentrations of free Ca2+ (0, 27.9, 34.9, 43.2, 52.5, 62.6, 131.2, 253.3, 377.3, and 501.0 μM) in 30 mM MOPS (pH 7.2), 100 mM KCl and 10 mM EGTA. To achieve precise concentrations of free Ca2+ in the micromolar range, we used solutions from the Calcium Calibration Buffer Kit #1 (Thermo Fisher Scientific). The MtSEO‐F1/Ca2+ mixtures were loaded into standard treated glass capillaries (NanoTemper Technologies) for measurement at 20°C, 20% LED intensity, and 20% laser intensity using a Monolith NT.115 (NanoTemper Technologies).
7
Bacterial Expression and Purification of GCaMP Variants
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Example 6
pRSET-A plasmids containing GCaMP variants were used to express protein in T7 Express E. coli cells (New England Biolabs) using 100 mL of ZYM-5052 auto-induction media and ampicillin at 30° C. for 48 h. Cells were lysed in B-PER (Thermo Scientific), 1 mg/mL lysozyme, 15 U/mL DNase at 22° C. for 30 min. After clearing, variants were purified using Ni2+-charged Profinity IMAC resin (Bio-Rad). Columns were washed with 20 mM Tris pH 8, 300 mM NaCl, 1 mM imidazole followed and then with 20 mM Tris pH 8, 500 mM NaCl, 10 mM imidazole. Variants were eluted in with 20 mM Tris pH 8, 100 mM NaCl, and 100 mM imidazole. Eluted protein concentrations ranged from 9-67 μM. Eleven-point calcium titrations were done using EGTA-buffered Ca2+ solutions, similar to the protocol of the Calcium Calibration Buffer Kit #1 (Life Technologies). Green fluorescence intensities (excitation 485 nm, 5 nm bandpass; emission 510 nm, 5 nm bandpass) were measured using a Safire2 plate reader (Tecan).
8
Bacterial Expression and Purification of GCaMP Variants
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Example 6
pRSET-A plasmids containing GCaMP variants were used to express protein in T7 Express E. coli cells (New England Biolabs) using 100 mL of ZYM-5052 auto-induction media and ampicillin at 30° C. for 48 h. Cells were lysed in B-PER (Thermo Scientific), 1 mg/mL lysozyme, 15 U/mL DNase at 22° C. for 30 min. After clearing, variants were purified using Ni2+-charged Profinity IMAC resin (Bio-Rad). Columns were washed with 20 mM Tris pH 8, 300 mM NaCl, 1 mM imidazole followed and then with 20 mM Tris pH 8, 500 mM NaCl, 10 mM imidazole. Variants were eluted in with 20 mM Tris pH 8, 100 mM NaCl, and 100 mM imidazole. Eluted protein concentrations ranged from 9-67 μM. Eleven-point calcium titrations were done using EGTA-buffered Ca2+ solutions, similar to the protocol of the Calcium Calibration Buffer Kit #1 (Life Technologies). Green fluorescence intensities (excitation 485 nm, 5 nm bandpass; emission 510 nm, 5 nm bandpass) were measured using a Safire2 plate reader (Tecan).
9
Coelenterazine Calcium Signaling Assays
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Coelenterazine (CTZ) for in vitro (#303) and in vivo (#3031) experiments was purchased from Nanolight Technologies. The Calcium Calibration Buffer Kit #1 from Life Technologies was used to prepare Zero Ca2+ buffer and 39 μM Ca2+ buffer. All other chemicals, including Ionomycin and Histamine, were purchased from Sigma.
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