Cells were cultivated in 96-well plates, 1 105 cells/well. A 5 μL of virus was added to cells with TCID50 being 107.4 and MOI being 1. Cells were grown in 96-well plates following CPV synchronous inoculation and 2.5% FBS medium cultivation for 48 h. DMSO was used to dissolve Fura-2 AM (catalog number: S1052, Beyotime Biotechnology Co., Ltd.) at a final concentration of 1 mM. Cell medium was replaced by 100 μL cell culture medium containing 5 μL Fura-2 AM per well following incubation at 37°C, 5% CO2 for 30 min. Calcium level was detected using a multifunctional microplate reader (Synergy ™ H4, BioTek), where the excitation and emission wavelengths were set at 340 nm and 510 nm, respectively. Data were analyzed using Gene5 software. Each sample has five replicates.
Fura 2 am
Manufactured by Beyotime
Sourced in China, United States
Fura-2 AM is a fluorescent calcium indicator used for the measurement of intracellular calcium concentrations. It is a cell-permeable acetoxymethyl (AM) ester derivative of the calcium-sensitive dye Fura-2. Upon entering cells, Fura-2 AM is hydrolyzed by intracellular esterases, releasing the active Fura-2 dye, which can then bind to calcium ions.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions)
Fluo 3 am, by Beyotime (2 mentions)
Pluronic f 127, by Beyotime (2 mentions)
Fura 2, by Zeiss (2 mentions)
Fluo 4 am, by Beyotime (2 mentions)
Fura 2 am, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Dm4000b, by Leica (1 mentions)
Bovine serum albumin (bsa), by BD (1 mentions)
Glutamate, by Merck Group (1 mentions)
Heat inactivated fetal calf serum, by Thermo Fisher Scientific (1 mentions)
Anti gadph, by Cell Signaling Technology (1 mentions)
Anti grp78, by Cell Signaling Technology (1 mentions)
Anti caspase 12, by Cell Signaling Technology (1 mentions)
Non fat milk powder, by BD (1 mentions)
Anti camkii, by Cell Signaling Technology (1 mentions)
Anti gadd153, by Cell Signaling Technology (1 mentions)
Anti xbp1, by Cell Signaling Technology (1 mentions)
Anti pcreb, by Cell Signaling Technology (1 mentions)
39 protocols using fura 2 am
1
Calcium Signaling Dynamics in Virus-Infected Cells
2
Measurement of Intracellular Calcium
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reshly prepared cardiac cryostat slides were incubated with the calcium indicator 10 μmol/L Fura‐2/AM (Beyotime) at room temperature in a humidified dark chamber for 30 min. After PBS washing for three times, samples were exited at 340 nm, and the fluorescence of Fura‐2/AM was observed at 510 nm with an inverted fluorescence microscope. Quantification of [Ca2+]i was presented as mean fluorescent intensity. For isolated myocytes, the cells were incubated with 5 μmol/L Fura‐2/AM (Thermo) at 37°C for 30 min in dark. The fluorescent intensity of Fura‐2/AM was detected by a flow cytometer (BD).
3
Intracellular Ca2+ Measurement in HaCaT Cells
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Fura-2 AM (Calcium ion fluorescent probes) (Beyotime，China) was used for the measurement of intracellular Ca2+ levels. HaCaT cells were seeded into 96-well plates at a density of 1 × 104 cells/well and incubated for 24 h at 37 °C and 5 % CO2. Next, HaCaT cells were washed twice with Krebs-HEPES buffer (120 mM NaCl, 5.4 mM KCl, 1.5 mM CaCl2, 1 mM NaH2PO4 and 10 mM HEPES (pH 7.4)) and subsequently incubated with 2 μM Fura-2 AM at 37 °C for 1 h. Then, HaCaT cells were washed twice with Krebs-HEPES buffer and treated with 0，50，100 and 200 μM EUG and 10 μM CAP (MACKLIN, China) for 30 min. Fluorescence intensity was detected using a multimode microplate reader at an excitation/emission wavelength of 340nm/510 nm. Finally, the results were visualized as fluorescence changes relative to an untreated control, expressed as (F–F0)/F0, where F and F0 represented the fluorescence intensity of the samples treated with EUG and CAP, and the untreated control, respectively.
4
Fluorescent Calcium Imaging in Seedlings
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The Ca2+ content was tested using the probe fluorescein Fluo-2-acetoxymethyl ester (Fura-2 AM) (S1052, Beyotime). The 4-day-old seedlings were stained with 0.5 µM Fura-2 AM for 45 min at room temperature (25°C), and washed with fresh liquid media three times before observation. All the experiments were captured for six different fields. The images were taken by fluorescence microscope (Leica DM4000B, Germany).
5
Longistyline C Isolation and Cell Assay
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Longistyline C was isolated from the leaves of pigeonpea by our laboratory [18 ] and the purity is over 95% which was detected by high performance liquid chromatography (HPLC) method.
Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY), penicillin (100 IU/ml Sigma, St. Louis, MO), streptomycin (10 μg/ml; Sigma), 10% heat-inactivated fetal calf serum (Gibco, Grand Island, NY). PC12 cells were purchased from the Cell Bank of Peking Union Medical College (Beijing, China). Methyl thiazolyl tetrazolium (MTT), dimethylsulfoxide (DMSO) and glutamate were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA), Fura-2/AM (Beyotime Institute of Biotechnology, Jingsu, China). Antibodies used for western blot analysis, primary antibodies: 1:1000 Anti-GADPH, 1:200 Anti-ERK1/2, 1:200 Anti-β-Actin, 1:100 Anti-GRP78, 1:200 Anti-p-ERk1/2,1:250 Anti-Caspase-12, 1:100 Anti-GADD153, 1:200 Anti-XBP-1, 1:500 Anti-Caspase-9, 1:1000 Anti-NMDAR/NR2B, 1:1000 Anti-CaMKII, 1:1000 Anti-CREB and 1:1000 Anti-p-CREB (Cell signaling Technology, USA); secondary antibodies: 1:1000 Anti-mouse-HRP and 1:1000 Anti-rabbit-HRP (Santa Cruz biotechnology, USA); Nonfat milk powder and bovine serum albumin (Becton, Dickinson and Company, USA). Double distilled water was used in all experiments. All the reagents were of analytical grade.
Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY), penicillin (100 IU/ml Sigma, St. Louis, MO), streptomycin (10 μg/ml; Sigma), 10% heat-inactivated fetal calf serum (Gibco, Grand Island, NY). PC12 cells were purchased from the Cell Bank of Peking Union Medical College (Beijing, China). Methyl thiazolyl tetrazolium (MTT), dimethylsulfoxide (DMSO) and glutamate were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA), Fura-2/AM (Beyotime Institute of Biotechnology, Jingsu, China). Antibodies used for western blot analysis, primary antibodies: 1:1000 Anti-GADPH, 1:200 Anti-ERK1/2, 1:200 Anti-β-Actin, 1:100 Anti-GRP78, 1:200 Anti-p-ERk1/2,1:250 Anti-Caspase-12, 1:100 Anti-GADD153, 1:200 Anti-XBP-1, 1:500 Anti-Caspase-9, 1:1000 Anti-NMDAR/NR2B, 1:1000 Anti-CaMKII, 1:1000 Anti-CREB and 1:1000 Anti-p-CREB (Cell signaling Technology, USA); secondary antibodies: 1:1000 Anti-mouse-HRP and 1:1000 Anti-rabbit-HRP (Santa Cruz biotechnology, USA); Nonfat milk powder and bovine serum albumin (Becton, Dickinson and Company, USA). Double distilled water was used in all experiments. All the reagents were of analytical grade.
6
Calcium Imaging in Primary Microglia
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The protocol for calcium imaging was performed as described (Hassan et al., 2014 (link)). The experiments were typically conducted using primary cultured microglia in 5 × 105 cells seeded onto 10 mm glass coverslips coated with PLL. Cultures were washed three times in buffer (145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 Glucose) and loaded for 30 min in the dark at 37°C, with 2 μM Ca2+ indicator dye, Fura-2 AM (Beyotime) and 1 μM cell-permeable Pluronic F-127 (Amresco). Cells were then washed three times with HBSS and incubated in extracellular solution at room temperature for 30 min before used in the dark. The intracellular calcium concentration was expressed as the F340/F380 ratios and the signals were captured and analyzed with NIS-Elements AR software (Nikon, Tokyo, Japan). Values were obtained from 150 to 200 cells in time-lapse images from each coverslip. Stimulating drugs were added after 2 min.
7
Intracellular Calcium and Metabolic Markers
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Cells were loaded with 2 μM Fura-2 AM (Beyotime, S1052) at 37 °C for 30 min, and intracellular calcium contents were determined by scanning with a microplate reader after washing. Intracellular ATP, GSH and NADPH levels and mitochondrial complex I activity were measured using commercial Kits. After treatment, the medium was collected for the assay of the released glutamate and lactate dehydrogenase (LDH) activity using commercial Kits.
8
Measuring Intracellular Calcium Dynamics in CTLs
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Intracellular calcium fluxes were measured as described previously (21 (link)). Briefly, CTLs with ASM or CD161 silencing were labeled with Fura-2-AM (Beyotime, #S1052, 1 μmol/L) for 30 minutes at 37°C. For CD161 cross-linking, T cells were incubated with anti-biotin microbeads (Miltenyi Biotec, #130-092-357) loaded with biotinylated anti-CD161 antibody (Miltenyi Biotec, #130-092-906) or isotype-biotin control (BioLegend, # 400104) for 3 minutes at 37°C. Intracellular Ca2+ was recorded at 500 nm emission in response to 340 nm/380 nm excitation using a fluorescence spectrophotometer (RF-5000; Shimadzu). T cells were stimulated by the addition of 40 μg/mL biotinylated anti-CD3ε (Miltenyi Biotec, #130-093-377, clone OKT3) followed by 20 μg/mL streptavidin (Thermo Fisher Scientific, #434301). For flow-cytometric analysis, T cells were labeled with Fura-3-AM (Beyotime, #S1056, 1 mmol/L) and Live/Dead Fixable Viability Dye-eFluor780. Calcium fluorescence was monitored at an emission wavelength of 530 nm with excitation at 488 nm. For fluorescent microscopy imaging, cells were loaded with Furo-3-AM, Hoechst 33342 (Thermo Fisher Scientific, # 62249), and PE-conjugated anti-human CD8a antibody (BioLegend, #301008) on a confocal dish and analyzed on an LSM 780.
9
Exosome-Mediated Cardioprotection Against Hypoxia
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The hiPSC-CMs were seeded onto 24-well plates or four-chamber slides and separately treated with PBS, EC-Exo (1 μg/mL), EC-ExoNC (1 μg/mL), EC-Exoanti−miR−100−5p (1 μg/mL), mimic NC (100 nM), and miR-100-5p mimic (100 nM). Mimic NC and miR-100-5p mimic were transfected using the Lipofectamine RNAiMAX Kit (Thermo Fisher Scientific, 13,778,075, USA). Then, cardiomyocytes were cultured under normal or oxygen–glucose deprivation conditions (glucose-free DMEM without serum, 1% O2/5% CO2/94% N2) for 48 h. Apoptosis was evaluated using an In Situ Cell Death Detection Kit (Roche Applied Science, 12,156,792,910, Germany). Lactate dehydrogenase (LDH) leakage in the culture medium was determined using a CytoTox-One homogenous membrane integrity assay (Promega, G7891, USA). The ATP content was measured in homogenized hiPSC-CMs using an ATP Bioluminescence Assay Kit (Sigma-Aldrich, 11,699,709,001, USA). The Ca2+ transients were evaluated by incubating the hiPSC-CMs with a Ca2+ indicator, Fura-2 AM (Beyotime, S1052, China), and electrically stimulating the cells. Then, the ratio of fluorescence emitted at 340 nm and 380 nm was recorded using a Ca2+-recording system (IonOptix, USA) [21 (link)].
10
Isolated Smooth Muscle Cell Protocol
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Mab was supplied by the Pharmaceutical Engineering Department, Shenyang Pharmaceutical University (enantiomeric excess >99%). Ach was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM) and Hanks' balanced salt solution (HBSS) were purchased from Gibco-BRL (Carlsbad, CA, USA) and type I collagenase from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). Fetal bovine serum (FBS) was produced by Tianjin Hualida Biotechnology Co., Ltd. (Tianjin, China). Triton X-100 and 3-(4,5-dimethylthinazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Amresco LLC (Solon, OH, USA). Mouse anti-α-smooth muscle actin (α-SMA), 5% bovine serum albumin (BSA), streptavidin-biotin complex (SABC) immunohistochemical staining kit and 3,3′ diaminobenzidine (DAB) chromogenic reagent kit were all purchased from Wuhan Boster Biological Technology Co., Ltd. (Wuhan, China). Fura-2/AM, Fluo-3/AM and fluorescein isothiocyanate (FITC)-labeled goat anti-mouse immunoglobulin G (IgG) were from Beyotime Institute of Biotechnology (Haimen, Jiangsu, China).
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