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Imaging of Intracellular Calcium in Hippocampal Slices: Methods, Limitations, and Achievements
Published in Avital Schurr, Benjamin M. Rigor, BRAIN SLICES in BASIC and CLINICAL RESEARCH, 2020
Menahem Segal, Jonathan M. Auerbach
Another fluorescent dye of the dual wavelength series is Indo-1. For both high and low [Ca2+]i, Indo-1 is excited by the same wavelength of light (355 nm).11 However, the emitted fluorescence measured at 405 nm increases and that at 490 nm decreases as a function of an increase in [Ca2+]i. For accurate spatial measurements of [Ca2+]i using this dye, one needs to take simultaneous images at the two wavelengths while verifying that these two images completely overlap. Advantages of this dye lie in the fact that the two images can be taken simultaneously, which is important in cases where one expects rapid changes in [Ca2+]i, and that there is no need for moving parts (i.e., filters) during the measurements since there is only one excitation wavelength.
Noninvasive Screening for Malignant Hyperthermia by Means of the Lymphocyte Test
Published in S. Tsuyoshi Ohnishi, Tomoko Ohnishi, Malignant Hyperthermia, 1994
Beverley A. Britt, Amira Klip, Peter J. O’Brien, Barbara I. Kalow
This potentiality has been resolved in studies using Indo 1 as the fluorescent indicator, since much lower intracellular concentrations of this indicator are required to obtain measurable fluorescence readings (see below).
Reciprocal SH2-SH3 Domain Contacts between ITK Molecules Limit T Cell Receptor Signaling in Th2-type CD4+ T Cells
Published in Immunological Investigations, 2022
Ji-Long Chen, Jennifer Y. Barr, Jonathan J. Zuk, Jacob V. Gorman, John D. Colgan
Cell were resuspended in stain buffer (PBS containing 3% FBS) and incubated with anti-mouse CD16/32 (eBioscience, San Diego, CA, USA) to prevent nonspecific antibody binding. Cells were incubated with fluorochrome-conjugated antibodies for 30 min on ice and then washed twice with stain buffer. Flow cytometric analysis was performed using an LSR II (BD Biosciences) and collected data analyzed using FlowJo (BD Biosciences). The fluorochrome-conjugated antibodies used were: PE-Cy7-CD4 (clone L3T4), PE-CD62L (clone MEL-14) and APC-B220 (RA3-6B2), all from eBioscience. To analyze calcium flux, Th2 cells were harvested after 5 days in culture, washed, and then rested for 5 days in medium containing 20 U/ml of IL-2. Cells were harvested and loaded with the dye Indo-1 (Invitrogen) by incubation at 37°C for 30 min followed by washing. Cells were then coated with biotinylated anti-CD3 (eBioscience) and washed to remove unbound antibody. Calcium flux was induced by adding streptavidin to 5 µg/ml (Sigma Aldrich).
Detecting T cell activation using a varying dimension Bayesian model
Published in Journal of Applied Statistics, 2018
Zicheng Hu, Jessica N Lancaster, Lauren I. R. Ehrlich, Peter Müller
Calcium flux, a increase in the intracellular calcium concentration, is a hallmark of T cell activation [19]. Therefore, it is often used as a proxy for T cell activation in biological assays. Intracellular calcium can be measured with a widely used ratiometric fluorescence calcium indicator, Indo-1 [7]. The emission peak of Indo-1 shifts from 475 to 405 nm upon Cain vitro assays where the activation of cells is highly synchronized and the excitation and emission light is not scattered by surrounding tissues [4,8].
Strong stimulation triggers full fusion exocytosis and very slow endocytosis of the small dense core granules in carotid glomus cells
Published in Journal of Neurogenetics, 2018
Amy Tse, Andy K. Lee, Noriko Takahashi, Alex Gong, Haruo Kasai, Frederick W. Tse
Indo-1 or indo1-FF was obtained from Teflabs (Austin, TX, USA). Nitrophenyl-EGTA (NP-EGTA) was a gift from Dr. Graham Ellis-Davis (Mount Sinai Hospital, NY, USA). All other chemicals were obtained from Sigma–Aldrich (Oakville, ON, Canada). The standard bath solution contained (in mM): 150 NaCl, 4.5 KCl, 2.5 CaCl2, 1 MgCl2, 8 glucose and 10 Na-HEPES (pH 7.4). The whole-cell pipette solution contained (in mM): 70 Cs-aspartate, 20 TEA-Cl, 40 Cs-HEPES, 1 MgCl2, 2 Na2ATP and 0.1 Na4GTP (pH 7.4).