Dissociated primary striatal neurons, or BV-2-derived microglia, were cultured on 35-mm glass-bottom dishes (MatTek, Ashland, MA) and were loaded with fura-2 AM (2.5 μm, Invitrogen, Carlsbad, CA) for 20 min (37°C, 5% CO2). Cells were then washed once and incubated in the medium for another 20 min to allow the de-esterification of the acetoxy methylester (AM) group. After that, the culturing dish was transferred to an automated, computer-controlled stage embedded on the Zeiss Axio Observer Z1 microscope and imaged using the physiology module of the Zeiss Zen software (Carl Zeiss Microscopy, LLC, Thornwood, NY). A series of fluorescent images (excitation at 340 and 380 nm, emission at 510 nm) were taken using an AxioCam MRm digital camera (Carl Zeiss Microscopy, LLC, Thornwood, NY) at a frame rate of 1 Hz during the first 90 s, 0.2 Hz during the next 60 s, 0.033 Hz from 2.5 min to 10 min, and 0.0166 Hz from 10 to 20 min. For each cell, 3 regions of interest (ROIs) were randomly selected in the cytoplasm. [Ca2+]i of each cell was calculated from the average F340/F380 ratio (Grynkiewicz et al, 1985 (link)) of 3 ROIs, using a standard curve generated by a calcium calibration buffer kit (Life Technologies, Carlsbad, CA).
Calcium calibration buffer kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Calcium Calibration Buffer Kit is a laboratory product designed to be used for the calibration and standardization of calcium measurement instruments. The kit includes a set of pre-formulated buffers with known calcium concentrations, enabling users to verify the accuracy and precision of their calcium analysis methods.
Automatically generated - may contain errors
Lab products found in correlation
Fura 2 am, by Thermo Fisher Scientific (1 mentions)
35 mm glass bottom dishes, by MatTek (1 mentions)
Zen software, by Zeiss (1 mentions)
Axiocam mrm digital camera, by Zeiss (1 mentions)
Axio observer z1 microscope, by Zeiss (1 mentions)
Axiovision rel 4, by Zeiss (1 mentions)
Pluronic f 127, by Thermo Fisher Scientific (1 mentions)
Fura 2 am ester, by Thermo Fisher Scientific (1 mentions)
Fp 8300 spectrofluorometer, by Jasco (1 mentions)
Avance 3 300 mhz spectrometer, by Bruker (1 mentions)
Nis elements ar 4, by Nikon (1 mentions)
Eclipse ti microscope, by Nikon (1 mentions)
Orbitrap fusion lumos tribrid, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Merck Group (1 mentions)
Lichropur, by Merck Group (1 mentions)
Chloroacetamide, by Merck Group (1 mentions)
Easy nlc 1200 system, by Thermo Fisher Scientific (1 mentions)
Methanol for hplc, by Merck Group (1 mentions)
Oregon green bapta 488 1 dextran, by Thermo Fisher Scientific (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
13 protocols using calcium calibration buffer kit
1
Calcium Imaging of Neuronal Cultures
2
Intracellular Calcium Measurement in Oligodendrocytes
3
Measuring Intracellular Calcium Dynamics
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Neurons were transfected with GCaMP2-encoding vectors (targeted to various locations), the fluorescence signal of which was detected using a standard GFP filter set (ex 480 ± 20; em 527 ± 15). Changes in Ca2+ were expressed as (F − Fmin) / (Fmax − F) according to the equation [Ca2+] = Kd*(F − Fmin) / (Fmax− F). Fmax was obtained when cells were treated with the cell-permeable Ca2+ ionophore ionomycin which both inserts into the plasma membrane and passes into the cell, inserting into mitochondrial membranes [75] (link), leading to saturation of the indicator when in regular medium (2 mM Ca2+). Fmin was obtained under the same conditions except in zero Ca2+ medium. The linear relationship between [Ca2+] and (F − Fmin) / (Fmax − F) has been previously confirmed by calibrating the indicator as expressed in neurons, exposing them to ionomycin in the presence of sequentially different solutions of precise [Ca2+], obtained by mixing K2EGTA and CaEGTA solutions (Calcium Calibration Buffer Kit, Invitrogen) at different ratios [82] (link).
4
Spectroscopic Characterization of Fluorescent Dyes
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All the solvents were of analytical grade. Chemicals were purchased from commercial sources. 1H-NMR and 13C-NMR were measured on a Bruker Avance III-300 MHz spectrometer (Bruker Biospin, The Woodlands, TX, USA) with chemical shifts reported in ppm (TMS as internal standard). Mass spectra were measured on a Focus GC/DSQ II spectrometer (ThermoScientific, Waltham, MA, USA) for IC and an API 3000 spectrometer (Applied Biosystems, PE Sciex) for ES. All pH measurements were made with a Mettler Toledo pH meter. Fluorescence spectra were recorded on a JASCO FP-8300 spectrofluorometer (JASCO, Easton, MD, USA). Absorption spectra were determined on a VARIAN CARY 300 Bio UV-Visible spectrophotometer. All measurements were done at a temperature of 25°C. The purity of the dyes were checked by RP-HPLC C-18, elutant: ACN 0.1% TFA/Water 0.1% TFA, method: 20/80 to 100/0 within 20 min then 100/0 for 10 min detection at λAbs = 254 nm. The apparent dissociation constant for calcium (KD Ca2+) was measured with a calcium calibration buffer kit from Invitrogen (Lifetechnologies, USA). All mass spectra, NMR spectra and chromatograms are included as supplemental data.
5
Quantifying Mitochondrial Calcium Dynamics
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This was performed essentially as described [66 ]. Neurons were transfected (Lipofectamine 2000) as previously described [67 (link)] with a mito-GCaMP2-encoding vector [68 (link)], the fluorescence signal of which was detected using a standard GFP filter set (ex 480±20; em 527±15). Changes in Ca2+ were expressed as (F-Fmin)/(Fmax-F) according to the equation [Ca2+] = Kd*(F-Fmin)/(Fmax-F). Fmax was obtained when cells were treated with the cell-permeable Ca2+ ionophore ionomycin which both inserts into the plasma membrane and passes into the cell, inserting into mitochondrial membranes, leading to saturation of the indicator when in regular medium (2 mM Ca2+). Fmin was obtained under the same conditions except in zero Ca2+ medium. The linear relationship between [Ca2+] and (F-Fmin)/(Fmax-F) has been previously confirmed by calibrating the indicator as expressed in neurons, exposing them to ionomycin in the presence of sequentially different solutions of precise [Ca2+], obtained by mixing K2EGTA and CaEGTA solutions (Calcium Calibration Buffer Kit, Invitrogen) at different ratios [8 (link)].
6
Intracellular Calcium Imaging in hESC-CMs
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Intracellular calcium imaging was performed as previously described [25] (link). Briefly, hESC-CMs were treated with 2 μM Fura-2AM (Fura-2 acetoxymethyl ester fura-2 AM-acetoxy-methyl ester) and cells were incubated for about 30 min at 37 °C in a dark room. After washing with HEPES, glass coverslips were placed on the recording chamber with HEPES buffer at a flow rate of 1.5 ml/min at room temperature. Images were recorded using an upright NIKON ECLIPSE Ti microscope equipped with a ratio metric imaging system (Nikon NIS-Elements AR 4.00.00). The ratio of 340 nm/380 nm fluorescence intensity (R340/380) within a certain region of interest after background subtraction was used as a relative measure of intracellular calcium concentration ([Ca2+]i) [26] (link). Calibration with external standards (Calcium Calibration Buffer Kit, Invitrogen) showed that R340/380 increased linearly with [Ca2+]i up to about 1 lM and R340/380 of 0.7–1.25 corresponded to basal [Ca2+]i of 90–180 nM. Therefore, the hESC-CMs with R340/380 in the range 0.7–1.25 were included in this study. All reagents were dissolved in HEPES buffer and applied locally to the hESC-CMs through a micropipette (with a tip diameter of 100 μm) and an 8-channel pressure-controlled drug application system (VC3-8PP, ALA Scientific).
7
Proteome Analysis by LC-MS/MS with TCEP and Trypsin
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Calcium calibration buffer kit, Invitrogen cat. no. C3008MP
Chloroacetamide, Sigma cat. no. C0267
TCEP, Sigma-Aldrich cat. no. C4706-2G
Rappsilbers Stage tipping Paper (23), C-18 material: CDS Empore C18 Extraction Disks, Fisher cat. no. 13-110-016
Formic acid for HPLC LiChropur, Sigma cat. no. 5438040100
Trifluoroacetic acid (TFA), HPLC Grade, 99.5+%, Alfa Aesar, cat. no. AA446305Y
Acetonitrile (ACN), Sigma-Aldrich cat. no. 271004-100 ML
Mass spectrometer: LC-MS: Orbitrap Fusion Lumos Tribrid (Thermo Fisher Scientific) and EASY-nLC 1200 System (Thermo Fisher Scientific)
Triethylammonium bicarbonate buffer 1.0 M, pH 8.5 ± 0.1, Sigma cat. no. T7408
Ethanol for HPLC, Sigma cat. no. 459828
Promega trypsin, cat no. V5111
Methanol for HPLC, Sigma cat. No. 494291
8
Calcium Imaging in Transfected SH-SY5Y Cells
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SH-SY5Y cells were transfected with constructs for 48 h by Lipofectamine (Thermo Fisher Scientific) following the manufacturer’s instructions. After 48 h, transfected SH-SY5Y cells were plated onto glass coverslips coated with poly-D-lysine at density of 10,000 cells/cm2. SH-SY5Y cells were loaded with Oregon Green 488 BAPTA-1 dextran (Invitrogen, 100 μg/ml) for 24 h. For staining of lysosome, CellLight lysosomes (LAMP-1)-RFP (Invitrogen) was also loaded to the SH-SY5Y cells. In vitro calcium-binding (Kd) affinities of Oregon Green 488 BAPTA-1 were determined using calcium calibration buffer kit (Invitrogen) adjusted to pH 4.5. The calibration curve was obtained as described previously [26 (link)]. In vitro minimal and maximal fluorescence (Fmin and Fmax) were calculated by perfusing the ionomycin, nigercin, and valinomycin pre-treated SH-SY5Y cells with 0 or 10 mM Ca2+ external solutions. The equation: [Ca2+]ly = Kd × (F-Fmin)/(Fmax-F) were used for calculation.
9
Fura-2 Calcium Imaging of RIN14B Cells
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RIN14B cells were placed on coverslips coated with poly-D-lysine and cultured for 24 h. Cells were then incubated with 10 µM fura-2 acetoxymethylester (Dojindo, Kumamoto, Japan) and 0.002% cremophor EL (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at room temperature in normal solution (140 mM NaCl, 3.3 mM KH2PO4, 0.8 mM K2HPO4, 1.2 mM CaCl2, 1.2 mM MgCl2, 10 mM glucose, and 20 mM HEPES, pH adjusted to 7.4 with NaOH). Fura-2 fluorescence was measured using an inverted microscope (Diaphot 300, Nikon, Tokyo, Japan) with a fluorescence ratio imaging system (ORCA-ER, Hamamatsu Photonics, Shizuoka, Japan). Cells were continuously superfused with normal solution and illuminated at 340 and 380 nm for 61.1 ms at 5 s intervals. The respective fluorescence signals (F340 and F380) were detected at 500 nm.
The intracellular calcium concentration ([Ca 2+ ]i) was calculated using a Calcium Calibration Buffer Kit (Invitrogen/Life Technologies). All experiments were performed at room temperature (22-25°C).
The intracellular calcium concentration ([Ca 2+ ]i) was calculated using a Calcium Calibration Buffer Kit (Invitrogen/Life Technologies). All experiments were performed at room temperature (22-25°C).
10
Protein labeling and Ca2+ binding assay
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6xHis-tagged protein was purified as described above and covalently labeled with an amine reactive fluorescent dye (MO-L001 Monolith™ Protein Labeling Kit RED-NHS) according to the manufacturer’s guidelines. Protein concentration was determined after labeling using the Pierce™ BCA Protein Assay Kit (Thermo Fischer Scientific) according to the manufacturer’s protocol. The final concentration of protein during the measurements was in the range of 30 nM. To ensure reliable concentrations of free Ca2+ in the assay mixture the assay buffers were formulated based on the Calcium Calibration Buffer Kit (Thermo Fischer Scientific) using an EGTA-Ca2+ system. The labeled proteins were measured at 15 different Ca2+ concentrations at an excitation energy of 40% and microscale thermophoresis power of 40% in the Monolith NT.115 (Nanotemper Technologies) using hydrophobic capillaries (MO-K003 Monolith™ NT.115 Hydrophobic Capillaries). The displayed analysis is based on three experimental replicates and analyzed using MO-S002 MO.Affinity Analysis software provided by the manufacturer (Nanotemper Technologies). For the Kd fit only the initial concentration of the labeled protein was provided (30 nM).
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