Flow cytometry was used to measure intracellular Ca2+ levels using Fluo-4 AM (Beyotime, Shanghai, China), and MDBK cells were incubated with Fluo4 AM at 37°C for 40 min (evading the light) after digesting and washing 3 times with Hanks balanced salt solution (HBSS). Subsequently, HBSS was added in 5-fold volume with 1% fetal bovine serum, and the cells were incubated at 37°C for an additional 40 min before being washed 3 times with HBSS. In each of the groups, approximately 10,000 or 20,000 cells were detected using a FACSCalibur flow cytometer (BD Biosciences) and analyzed with FlowJo software (version 10.0.7).
Fluo 4 am
Manufactured by Beyotime
Sourced in China, United States
Fluo-4 AM is a calcium indicator dye that can be used to measure intracellular calcium levels in living cells. It is a cell-permeant acetoxymethyl (AM) ester form of the fluorescent indicator Fluo-4.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Reactive oxygen species assay kit, by Beyotime (4 mentions)
Fluo 4 am, by Zeiss (3 mentions)
Dcfh da, by Beyotime (3 mentions)
Poly d lysine hydrobromide pdl, by Merck Group (3 mentions)
Triton x 100, by Avantor (3 mentions)
Penicillin, by Beyotime (3 mentions)
Streptomycin, by Beyotime (3 mentions)
Lipid peroxidation mda assay kit, by Beyotime (3 mentions)
Cytoflex, by Beckman Coulter (3 mentions)
Fv1000, by Olympus (3 mentions)
Facscalibur, by BD (3 mentions)
Pluronic f 127, by Thermo Fisher Scientific (3 mentions)
H2dcfda, by Thermo Fisher Scientific (3 mentions)
Er tracker red, by Beyotime (2 mentions)
Fluo 4 am, by Beckman Coulter (2 mentions)
Pluronic f 127, by Beyotime (2 mentions)
Confocal microscope, by Leica (2 mentions)
Fluorescence microscopy, by Zeiss (2 mentions)
Ldh cytotoxicity assay kit, by Beyotime (2 mentions)
156 protocols using fluo 4 am
1
Intracellular Calcium Measurement by Flow Cytometry
2
Intracellular Ca2+ Dynamics in HUVECs
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HUVECs were seeded at a density of 1×105 cells/well in 12-well plate for 24 h. Then, HUVECs were treated with the corresponding stimuli for a period of time, and washed 3 times with a Ca2+-free D-Hanks balanced salt solution. Subsequently, the cells were loaded with Fluo-4AM (Beyotime, S1060) for 1 h at 37℃ in dark, then washed 2 times with Ca2+-free D-Hanks balanced salt solution to remove extracellular Fluo-4-AM. Therefore, the fluorescence intensity was quantitatively analyzed using a microplate reader (BioTek, USA), and qualitatively detected using a Nikon ECLIPSE Ti microscope (Nikon, Japan).
3
Calcium Imaging in Hippocampal Neurons
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Scanning laser confocal microscopy (Leica TCS SP2, Wetzlar, Germany) was used to determine cytosolic Ca2+ in primary cultured hippocampal neurons at day in vitro 12 (DIV12). The cells were loaded with 5 μM Fluo-4/AM (Beyotime, Shanghai, China) in Hank’s solution at 37 °C in the dark for 45 min, then washed using Hank’s solution gently to remove extracellular Fluo-4/AM dye. Fluorescence was monitored immediately every 10 s over a period of 15 min (excitation at 488 nm and emission at 526 nm). Prior to NMDA stimulation, the dye-loaded cells were scanned for ~2 min to obtain a basal level of fluorescence intensity. After different cell treatments as mentioned above, the fluorescence intensity was monitored and the dynamic change of the intracellular Ca2+ concentration ([Ca2+]i) was recorded. The [Ca2+]i value was measured according to the relative fluorescence intensity to the basal level.
4
Fluo-4 AM Calcium Imaging Protocol
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Following 3 washes with D-PBS, 0.3 mM Fluo-4 AM (Beyotime) was added to the cells for 40 min at 37 °C, and then, the cells were washed with D-PBS for 3 times. The cells were incubated at 37 °C for 20 min before observation with A1R confocal microscope (Nikon, Japan). Videos were captured in continuous shooting mode using 488 channels.
5
Intracellular Calcium Measurement in MC3T3-E1 Cells
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The concentration of intracellular Ca2+ in the MC3T3-E1 cells was examined using Fluo-4 AM (Beyotime Institute of Biotechnology). The cells inoculated in 24-well plates were washed three times with PBS to ensure that the medium was completely removed. Subsequently, the cells were completely covered with 300 µl Fluo-4 AM working solution and incubated at 37°C for 30 min. After washing twice with PBS, the cells were incubated for a further 30 min to ensure that the Fluo-4 AM in the cells was completely transformed into Fluo-4 with fluorescence. The fluorescence intensity of intracellular Fluo-4 was measured, which represented the concentration of Ca2+. Bay K8644 (Dalian Meilun Biology Technology Co., Ltd.), as a highly selective L-type calcium channel agonist, was used to increase cytoplasmic calcium concentration. Bay K8644 at a concentration of 10 µM was added to the MC3T3-E1 cells with metformin and incubated for 48 h at 37°C.
6
Melatonin Effects on Intracellular Calcium
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MC3T3-E1 cells were cultured in a 6-well plate at a density of 2×105 cells/well. The cells were cultured with or without 100 nM or 10 μM melatonin. After 24 h of culture, the supernatant was removed, and the cells were washed twice with PBS and then digested with 0.25% trypsin. Digestion was stopped by adding α-MEM supplemented with 10% FBS. The cells were suspended and centrifuged (1000 rpm for 5 min). The supernatant was removed, and the cells were washed twice with PBS and centrifuged again to obtain cell pellets. Subsequently, the cells were stained with 200 μL of Fluo-4/AM (Beyotime, Shanghai, China) (5 μmol/L) and then incubated at 37°C for 30 min. Finally, the cells were washed three times with PBS. The results were quantified using a full-wavelength multifunctional microplate reader at an excitation wavelength of 490 nm and an emission wavelength of 525 nm.
7
Immunofluorescence Staining of Organelles
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Mouse monoclonal anti-Rab6a (Cat# ab55660) and GM130 antibody (Cat# ab52649) were purchased from Abcam (Cambridge, MA, USA); Mouse monoclonal anti-α-tubulin-FITC antibody (Cat# 76074), Phalloidin-FITC (Cat# P5282) and Lectin-FITC (Cat# L4265) were purchased from Sigma (St. Louis, MO, USA); FITC-conjugated goat anti-rabbit IgG was purchased from Thermo Fisher Scientific (Rockford, IL, USA). ER-tracker red (Cat# C1041-1) was purchased from Beyotime, Fluo-4 AM (Cat# F14201), and Pluronic F-127 (Cat# P-3000MP) were purchased from Thermo Fisher Scientific.
8
Intracellular Calcium Dynamics Assay
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Cells were incubated for 30 min in the presence of 0.5 μM fluo-4 AM (Beyotime, S1060), a Ca2+ indicator dye, then washed twice with Hanks’ balanced saline solution and allowed to incubate for 20 min before detection. The fluorescence intensity of intracellular Ca2+ was monitored with a microplate reader prior to and following exposure to 1 μM thapsigargin (Sigma-Aldrich, T9033).
9
Calcium Influx Measurement in Cell Lines
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A172 and LN229 were seeded onto six-well plates. After 24 hours, cells were harvested with 2 μM of Fluo-4 AM for 30min at 37℃ according to manufacturer's recommendations (Beyotime, China).
Washed with PBS without calcium three times, cells were treated with or without UDCA for 30min followed by fluorescence imaging. Mean optical intensity was calculated by dividing the integrated intensity by the area of the measured spot with NIH imageJ software.
Washed with PBS without calcium three times, cells were treated with or without UDCA for 30min followed by fluorescence imaging. Mean optical intensity was calculated by dividing the integrated intensity by the area of the measured spot with NIH imageJ software.
10
Intracellular Calcium Measurement using Fluo-4 AM
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The intracellular calcium concentration was measured with Fluo-4AM (Shanghai Beyotime Bio-Tech Co., Ltd.) according to manufacturer's instructions. In detail, cells were washed twice with phosphate-buffered saline (PBS) and digested with trypsin for 5 min at 37°C. An equal quantity of DMEM supplemented with 10% FBS was added to terminate digestion. The cells were centrifuged at 1,000 rpm for 5 min, the supernatant was removed, and the cells were washed once with PBS and centrifuged again to obtain a cell pellet. Subsequently, the cells were stained with 200 µL of Fluo-4 AM ( nal concentration of 5 µM), incubated for 30 min in PBS at 37°C, washed three times with PBS and incubated in PBS for an additional 15 min in the absence of Fluo-4AM to allow complete de-esteri cation of the dye. The uorescence intensity was obtained by ow cytometry with 488-nm laser excitation and a 512-520-nm emission lter.
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