Two days after plating 2.5 × 104 H295R cells into 96-well plates, the cells were incubated with and without 10 mM YM750 for 24 h. The cells were then loaded with Fluo 4-AM (Dojindo, Rockville, MD, USA; 5 mg/mL) in the presence of 1.25 mM probenecid (Dojindo) and 0.04% Pluronic F-127 (Dojindo) for 1 h. Cells were then washed with PBS and the recording medium containing 1.25 mmol/L probenecid and 20 mM KCl was added to the media. Changes in intracellular calcium concentration were determined by measuring the fluorescence intensity (excitation wavelength, 485 nm; emission wavelength, 535 nm).
Pluronic f 127
Manufactured by Dojindo Laboratories
Sourced in Japan, United States
Pluronic F-127 is a non-ionic, triblock copolymer surfactant composed of polyethylene oxide (PEO) and polypropylene oxide (PPO) segments arranged in a PEO-PPO-PEO structure. It is a white, waxy solid at room temperature and has a low critical micelle concentration (CMC).
Automatically generated - may contain errors
Lab products found in correlation
Probenecid, by Dojindo Laboratories (5 mentions)
Fluo 4 am, by Dojindo Laboratories (4 mentions)
Lsm 510 meta confocal microscopy, by Zeiss (1 mentions)
Zen 2009, by Zeiss (1 mentions)
Prism 5, by GraphPad (1 mentions)
Fluo 4, by Dojindo Laboratories (1 mentions)
Celllux, by PerkinElmer (1 mentions)
Fura 2 am, by Merck Group (1 mentions)
Fura 2 am, by Dojindo Laboratories (1 mentions)
Fv1000, by Olympus (1 mentions)
Hanks balanced salt solution (hbss), by BD (1 mentions)
Hanks balanced salt solution (hbss), by Beyotime (1 mentions)
Fluo 3 am, by Dojindo Laboratories (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco s phosphate buffered saline, by Thermo Fisher Scientific (1 mentions)
Nifedipine, by Merck Group (1 mentions)
Human mcp 1 elisa kit, by USCN (1 mentions)
Glyburide, by Merck Group (1 mentions)
9 protocols using pluronic f 127
1
Intracellular Calcium Measurement in H295R Cells
2
Measuring Intracellular Calcium Dynamics
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Intracellular calcium assay was performed as previously described [30 (link)]. The culture medium of the Flp-In NCC HEK293 cells was replaced by loading buffer containing 5 μg/ml Fluo 4-AM (Dojindo, Kumamoto, Japan), 1.25 mmol/l probenecid (Dojindo, Kumamoto, Japan), and 0.02% Pluronic F-127 (Dojindo, Kumamoto, Japan). Following incubation with 5 mM EGTA or 1 μM SEA0400 in the loading buffer for 1h at 37°C, the loading buffer was replaced by recording medium containing 1.25 mmol/l probenecid. NCX1 siRNA silencing was applied 48 h before loading buffer replacement. Following the administration of KCl (final concentration: 10 mM), the fluorescence intensities of Fluo 4 were quantified from five regions of interest using LSM 510 Meta confocal microscopy and the Zen 2009 software (Carl Zeiss, Oberkochen, Germany).
http://dx.doi.org/10.17504/protocols.io.baihicb6
3
Analyzing hiPSC-CM Spheroid Responses
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Pure hiPSC-CM spheroids on day 5, after they started beating, were analyzed after replacing the medium with fresh medium and performing incubation at 37°C for 30 min. We exposed the spheroids to several compounds at different concentrations for 30 min and recorded the findings with a BZX-710. The beating rates were calculated based on moving images. We also detected the onset of the changes in beating after the administration of COA-Cl. We continuously observed the beating spheroids before and after the administration of COA-Cl using fluorescent calcium imaging. The spheroids were incubated for 1 h at 37°C in loading buffer containing 10 μM fluo4 acetoxymethyl ester (Dojindo, Kumamoto, Japan) and detergents (0.02% Pluronic F-127, and 1.25 mmol/l Probenecid) (Dojindo). The samples were washed with recording buffer (Dojindo) for 20 min at 37°C, and calcium transients were recorded using fluorescence imaging with an excitation wavelength of 488 nm. The videos were converted to a series of TIFF format pictures, and the relative fluorescence (in relative fluorescence units [RFU]) of the spheroids was measured using the BZ-X Analyzer software program. The measured RFU was graphed to determine the beating profiles of RFU with the Excel software program.
4
Wnt5a-Induced Calcium Signaling in mpkCCD
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The culture medium of the mpkCCD cells or dissection solution of mouse kidneys was replaced with loading buffer containing 5 μg ml−1 Fluo4-AM (Dojindo), 1.25 mmol l−1 probenecid (Dojindo), and 0.04% Pluronic F-127 (Dojindo). After incubation for 1 h at 37 °C, loading buffer was replaced with recording medium containing 1.25 mmol l−1 probenecid. The mpkCCD cells and isolated CCD of mouse kidneys were then placed under confocal laser scanning microscopy. Wnt5a (500 ng ml−1) was added, and calcium responses were monitored by the LSM510 Meta. Fluorescence intensities of Fluo4 were quantified from six regions of interest using Zen 2009 software.
5
Quantifying Receptor-Mediated Calcium Signaling
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Chinese hamster ovary (CHO)‐K1 cells expressing M1R–M5R were plated on a 96‐well black, clear bottom plate at 30 000 cells/well and incubated at 37°C in an atmosphere of 5% CO2 for 1 day. On the day of the assay, cells were incubated with calcium dye buffer (Hanks’ Balanced Salt Solution (HBSS) containing 20 mmol/L HEPES, 0.1% fatty acid‐free bovine serum albumin (BSA), 0.08% pluronic F127 (Dojindo Laboratories), 2.5 μg/mL Fluo‐4 (Dojindo Laboratories), and 1.25 mmol/L probenecid (Dojindo Laboratories)) for 30 minutes at 37°C in an atmosphere of 5% CO2 and then incubated for 30 minutes at room temperature. To measure Ca2+ mobilization using CellLux (PerkinElmer), cells were stimulated with T‐495 or MK‐7622 (0.01‐1000 nmol/L for PAM activity; 0.3‐10 000 nmol/L for agonist activity) in assay buffer (HBSS containing 20 mmol/L HEPES and 0.1% fatty acid‐free BSA) with or without an EC20 concentration of ACh. The inflection point (IP) and EC50 values were calculated using the following equation by GraphPad Prism 5 software (GraphPad Software Inc):
where X and Y are the log concentration of a compound and the percentage of Ca2+ response, respectively, and Top and Bottom are the upper and lower plateaus, respectively.
where X and Y are the log concentration of a compound and the percentage of Ca2+ response, respectively, and Top and Bottom are the upper and lower plateaus, respectively.
6
GABA-Induced Calcium Signaling in OUMS-27 Cells
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The intracellular Ca2+ concentration, [Ca2+]I, in drug-treated OUMS-27 cells, was measured by Fura-2 AM, a calcium-sensitive dye (Dojindo, Kumamoto, Japan). Briefly, OUMS-27 cells were loaded with 5 μM Fura-2 AM in loading buffer containing 0.01% pluronic F-127 (Dojindo) for 30 min at 37 °C. After washing Fura-2 AM out of the loading buffer, the relative transient calcium concentration (OD340nm/OD380nm excitation ratio) was recorded before and after the addition of 10 mM GABA (Sigma-Aldrich) in a perfusion chamber using the AQUACOSMOS/RATIO, C7773 (Hamamatsu photonics, Hamamatsu, Japan). Recording was continued after washing the 10 mM GABA out of the buffer. [Ca2+]I after pretreatment with 1 μM CGP and application of 10 mM GABA was recorded using a similar method.
7
Measuring PCV2-induced Cytosolic Calcium Changes
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To explore if PCV2 infection could perturb Ca2+ homeostasis of cells as part of the signaling components, cytosolic Ca2+ level in PCV2-infected PK-15 cells was measured by flow cytometric method. Cells were washed twice with Hanks’ balanced salt solution (HBSS, Beyotime), loaded with the Ca2+ indicator dye Fluo 3-AM (2.5 μM) and Pluronic F-127 (0.02%) (Dojindo, Shanghai, China) and incubated for 45 min at 37 °C. Cells were then washed twice and suspended in 1 mL HBSS for flow cytometry (Becton Dickinson, San Jose, CA, USA). Fluorescence intensity was collected at 488 nm (excitation) and 530 nm (emission) under the logarithmic mode. The fluorescence intensities of various treatments were expressed relative to those under mock conditions.
For analysis of cytosolic Ca2+ by FRET, the PCV2-infected cells were imaged using a confocal microscope. FRET efficiency was calculated with the acceptor photobleaching method according to the manufacturer’s instructions (FV1000; Olympus; Markham, ON, Canada).
For analysis of cytosolic Ca2+ by FRET, the PCV2-infected cells were imaged using a confocal microscope. FRET efficiency was calculated with the acceptor photobleaching method according to the manufacturer’s instructions (FV1000; Olympus; Markham, ON, Canada).
8
Investigating Macrophage Inflammatory Response Pathways
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DMEM, foetal bovine serum (FBS), Dulbecco's phosphate‐buffered saline (DPBS) and HEPES were purchased from Invitrogen (Burlington, ON, Canada). Human MCP‐1 ELISA Kit was from Uscn Life Science Inc. (Houston, TX, USA). Nifedipine, glyburide and dimethyl sulfoxide (DMSO) were obtained from Sigma‐Aldrich (St. Louis, MO, USA). Fluo‐4‐AM and Pluronic F‐127 were purchased from DOJINDO (Rockville, MD, USA). Antibodies recognizing phosphorylation of P‐ERK, P‐JNK, P‐p38 and P‐c‐jun were from Cell Signaling Technology (Danvers, MA, USA). Antibodies recognizing LOX‐1, SR‐AI and CD36 were purchased from Abcam (Cambridge, MA, USA). Inhibitors of ERK (PD98059), JNK (SP600125) and p38 (SB203580) were from Beyotime (Beijing, China). Antibodies of CD16, CD32 and CD64 were purchased from Santa Cruz Biotechnology Inc. (Dallas, Texas, USA). The oxLDL monoclonal antibody (BI‐204) and control antibody (FITC‐8) were kindly provided by BioInvent International AB (Lund, Sweden). Antibodies recognizing TLR‐4 and β‐actin were from Proteintech Group Inc. (Chicago, IL, USA). BCA protein assay reagents, BSA standards and SuperSignal Femto substrate were purchased from Pierce (Milwaukee, WI, USA).
9
Intracellular Ca2+ Imaging in Heart Organoids
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The heart organoids were washed two times with PBS, incubated at 37 °C for 15−30 min with 4 μM of the green fluorescent intracellular Ca2+-binding dye Fluo8-AM (AAT Bioquest, Sunnyvale, CA) with Pluronic F127 (Dojindo, Tokyo, Japan) in normal Tyrode’s solution, and then washed again two times with PBS. Subsequently, the heart organoids were mounted in glass-bottom dishes with 200 μl of normal Tyrode’s solution and observed under a fluorescence microscope (BZ-X710, Keyence). Isoproterenol was used in the final concentration of 1 μM. The obtained images were analyzed with ImageJ software. The time constant of Ca2+ decay was calculated by a single exponential fit using Origin software (OriginLab).
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