THP-1 cells were seeded at a density of 5x103 cells/well in 96-well plates and treated with 100 ng/ml PMA at 37˚C for 24 h to transform into adherent macrophages. Then the cells of different treatment groups were incubated with M.tb (MOI:35) and vitamin D3 (5, 10 and 20 µmol/ml) at 37˚C for 24 h. Cytosolic Ca2+ concentration was quantified using Fluo-4 acetoxymethyl (AM; Thermo Fisher Scientific, Inc.). In brief, Fluo-4 AM was diluted in PBS and the cells were incubated at a final concentration of 1 µM for 30 min at 37˚C to ensure that Fluo-4 AM was fully converted into Fluo-4 intracellularly. Cells were then washed three times with PBS solution, and the fluorescence intensity was assessed by acquiring emissions at 494 nm using flow cytometry. (Thermo Fisher Scientific, Inc.). FlowJo version 10 software (FlowJo, LLC) was used for flow analysis. The result was compared with the control group.
Fluo 4 acetoxymethyl am
Manufactured by Thermo Fisher Scientific
Sourced in United States
Fluo-4 acetoxymethyl (AM) is a fluorescent calcium indicator. It is a cell-permeable calcium-sensitive dye that can be used to measure intracellular calcium levels in live cells.
Automatically generated - may contain errors
Lab products found in correlation
Version 10, by FlowJo (1 mentions)
A23187, by Merck Group (1 mentions)
Alpha iκbα, by Cell Signaling Technology (1 mentions)
Dinitrophenyl dnp ige, by Merck Group (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide, by Merck Group (1 mentions)
α tubulin, by Santa Cruz Biotechnology (1 mentions)
Phospho p38, by Cell Signaling Technology (1 mentions)
Caspase 1 antibody, by Santa Cruz Biotechnology (1 mentions)
Evans blue, by Merck Group (1 mentions)
Phospho jnk, by Cell Signaling Technology (1 mentions)
Compound 48 80, by Merck Group (1 mentions)
Phospho nf κb, by Cell Signaling Technology (1 mentions)
Antibodies against phospho erk, by Cell Signaling Technology (1 mentions)
Lamin b, by Santa Cruz Biotechnology (1 mentions)
Nis elements ar analysis 4, by Nikon (1 mentions)
Eclipse ti2, by Nikon (1 mentions)
Glass bottom dishes, by MatTek (1 mentions)
Pluronic f 127, by Merck Group (1 mentions)
4 protocols using fluo 4 acetoxymethyl am
1
Vitamin D3 Modulates Calcium Signaling in Macrophages
2
Anti-inflammatory Effects of G-Rg3
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Prednisolone (PDN) and antibodies against phospho-ERK, phospho-JNK, phospho-p38, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα), and phospho-NF-κB were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-ERK, p38, JNK, α-tubulin, Lamin B, RIP2, and caspase-1 antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). G-Rg3 (CAS no. 14197-60-5, purity: 95–99%) was purchased from Chengdu Biopurify Phytochemical Ltd. (Chengdu, Sichuan, China). Water Soluble Tetrazolium Salt (WST) reagent (EZ-cytox) was purchased from DoGen (Seoul, Korea). Fluo-4 acetoxymethyl (AM) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). 3-(4 5-dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bromide (MTT), Compound 48/80, Evans blue, Dinitrophenyl (DNP)-IgE, DNP-Bovine Serum Albumin (BSA), phorbol 12-myristate 13-acetate (PMA), and A23187 were purchased from Sigma-Aldrich (St. Louis, MO, USA).
3
Confocal Imaging of Intracellular Calcium
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For confocal imaging of intracellular calcium (Ca2+), HBVPs were seeded on glass-bottom dishes (MatTek, Ashland, MA, USA). At 70% confluence, primary cells were treated with either DMSO or PGE2 (10 nm or 100 nm) for 24 and 72 h. Following treatment, cells were washed with Krebs–Ringer-HEPES buffer and incubated with Fluo-4 acetoxymethyl (AM) (5 mM; Invitrogen) for 40 min. After successive washes and incubation with KRH for 30 min, the level of intracellular Ca2+ in pericytes was assessed by basal Fluo-4 fluorescence imaging using a laser confocal microscope (Nikon Eclipse Ti2, Nikon Instruments, Melville, NY). Processing and analysis of the acquired images were carried out using Nikon NIS-Elements AR Analysis 4.40 software. Resting cytosolic Ca2+ levels were measured using the ROI measurement tool, considering each pericyte a single ROI in selected multi-regions of interest (multi-ROIs). Approximately 600–700 cells were scored for each experimental condition.
4
Fluo-4-AM Cytoplasmic Ca2+ Imaging
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Cytoplasmic free, ionized calcium level was detected by loading the cells with 5 μM Fluo-4- acetoxymethyl (AM) (Invitrogen, Thermo Fisher Scientific in DMSO, Sigma-Aldrich) supplemented with 20% (w/v) Pluronic F-127 (Sigma-Aldrich) for 15 minutes in the dark at RT. Cells were washed and then kept in RPMI medium supplemented with CaCl2 to a Ca2+ concentration of 1.8 mM. Cells were incubated in RT in the dark for a further 30 minutes to allow complete de-esterification of intracellular Fluo-4-AM esters. Each sample was measured directly after the de-esterification period.
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