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Indo 1 am

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Indo 1-AM is a fluorescent calcium indicator dye used for measuring intracellular calcium concentrations in living cells. It is a cell-permeant form of the indicator dye Indo-1, which undergoes a shift in its fluorescence emission spectrum upon binding to calcium ions.

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Lab products found in correlation

Infinite 200 pro microplate reader, by Tecan (1 mentions) Ter119, by BioXCell (1 mentions) Low melt agarose, by Lonza (1 mentions) Vt1000s vibratome, by Leica (1 mentions) Pluronic f 127, by Thermo Fisher Scientific (1 mentions) Fluostar omega, by BMG Labtech (1 mentions) Lsrii sorp, by BD (1 mentions)

5 protocols using indo 1 am

1

Calcium Content Quantification Using Indo 1-AM

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To quantify calcium content on each sample, a fluorescence assay using Indo 1-AM (I3261, Sigma-Aldrich) was performed following the manufacturer’s protocol. Briefly, each hydrogel or film sample was smashed and incubated with Indo 1-AM solution (10 µM, dissolved in dimethyl sulfoxide (DMSO)) for 30 min at 37 °C. After being centrifuged for 5 min at 12,000 rpm, the supernatant was collected, and the fluorescence emission spectra were measured at 355 nm by Infinite 200 Pro microplate reader (Tecan). The unreacted calcium ions were detected at 460 nm.
An Y.H, & Kim S.H. (2021). Facile Fabrication of Three-Dimensional Hydrogel Film with Complex Tissue Morphology. Bioengineering, 8(11), 164.
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2

Thymocyte Motility Imaging by Two-Photon Microscopy

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CD4SP thymocytes were enriched by magnetic depletion using antibodies against CD8, Gr-1, Ter119, B220, CD25, and CD11b (BioXCell). 1×106Ebi2+/+ and Ebi2-/- CD4SP cells were stained with Indo-1AM (Sigma) and CMTPX red (Life Technologies), respectively, for 30min at 37°C,according to manufacturers’ instructions and mixed at a 1:1 ratio in complete RPMI. 3-4 week old pCX-EGFP thymi were embedded in low-melt agarose (Lonza) and vibratome sectioned, with a VT1000S Vibratome (Leica), as previously described [12 (link)]. After incubating thymocytes on slices for 1-2 h at 37°C 5% CO2, two-photon images were acquired every 15 s, through a depth of 40 μm at 5-μm intervals using an Ultima IV microscope equipped with a 20x NA 1.0 water-immersion objective, and PraireView software (Prairie). MaiTai lasers (SpectraPhysics) tuned to 740 nm and 900 nm were used to excite Indo-1 and EGFP/CMTPX, and emitted light was passed through 400/50, 480/40, 535/50, and 607/45 filters (Chroma Technology) for detection of the two Indo1 emission peaks, EGFP, and CMTPX, respectively. were carried out with Imaris v.8 (Bitplane).
Ki S., Thyagarajan H.M., Hu Z., Lancaster J.N, & Ehrlich L.I. (2017). EBI2 contributes to the induction of thymic central tolerance by promoting rapid motility of medullary thymocytes. European journal of immunology, 47(11), 1906-1917.
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3

Monitoring CNGA1 channel activity

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The fluorescent indicator Indo-1/AM was used to monitor Ca2+ influx through the CNGA1 channels in cell suspensions. The assays were performed as described [10 (link),15 (link)] using a spectrofluorometer (Fluostar Omega, BMG lab tech GmBH, Offenburg, Germany). This assay was designed to determine CNG channel activity in cell populations (2 × 106) in response to 8-pCPT-cGMP stimulation. Briefly, cells (36–48 h post-transfection) were harvested with cell dissociation medium (Invitrogen, Carlsbad, CA), washed with the extracellular solution (ECS; 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 10 mM glucose, 15 mM HEPES, pH 7.4), and incubated with 2 μM Indo-1/AM (Sigma-Aldrich) in ECS in the presence of 0.05% Pluronic F-127 (Invitrogen, Carlsbad, CA) for 40 min at room temperature. Then, the cells were washed three times with ECS and resuspended in ECS (1 × 106/mL). Ca2+ influx in response to 8-pCPT-cGMP was determined by ratiometric measurement, which represents the free intracellular Ca2+ concentration. Changes of intracellular Ca2+ concentration were expressed as a ∆405/485 ratio.
Gupta V.K., Rajala A, & Rajala R.V. (2015). Non-canonical regulation of phosphatidylinositol 3-kinase gamma isoform activity in retinal rod photoreceptor cells. Cell Communication and Signaling : CCS, 13, 7.
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4

Melan-A-specific CD8 T-cell Calcium Flux

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Calcium mobilization assay was performed as described previously (37 (link)). Briefly, vaccine-induced Melan-A-specific CD8 T-cells were loaded with 2 μM Indo 1-AM (Sigma-Aldrich) for 1 h, washed and resuspended at a concentration of 106 cells in pre-warmed RPMI containing 2% of FCS. Baseline signal was recorded for 30 s before 1 μg/ml analog HLA-A2/Melan-A26−35-specific multimers were added and intracellular Ca2+ flux was assessed for 5 min under UV excitation and constant temperature (37°C) using a thermostat device on an LSR II SORP (BD Biosciences) flow cytometer. Indo-1 (violet)/Indo-1 (blue) 405/425 nm emission ratio was analyzed by the FlowJo kinetics module software (v.9.7.6, Tree Star).
Carretero-Iglesia L., Couturaud B., Baumgaertner P., Schmidt J., Maby-El Hajjami H., Speiser D.E., Hebeisen M, & Rufer N. (2020). High Peptide Dose Vaccination Promotes the Early Selection of Tumor Antigen-Specific CD8 T-Cells of Enhanced Functional Competence. Frontiers in Immunology, 10, 3016.
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5

Intracellular Calcium Measurement in VSMCs

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Indo 1-AM (Sigma, Switzerland) was used as a fluorescent indicator for the determination of intracellular calcium. VSMC were loaded with 2 mg/ml Indo 1-AM and incubated for 1 h at 37 C. Cells were then washed with DMEM, supplemented with secondary CPP for 30 and 60 min and subsequently analyzed on a FACS (LSR II) flow cytometer [4] (link). Data were analyzed using the FlowJo (Treestar) software.
Aghagolzadeh P., Bachtler M., Bijarnia R., Jackson C., Smith E.R., Odermatt A., Radpour R, & Pasch A. (2016). Calcification of vascular smooth muscle cells is induced by secondary calciprotein particles and enhanced by tumor necrosis factor-α. Atherosclerosis, 251.
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