The NRMCs were incubated with 1 μmol/L calcein acetoxymethyl ester (Sigma‐Aldrich, Cat. No. 56496 calcein‐AM, Sigma, St. Louis, MO) dissolved in dimethyl sulfoxide at room temperature for 30 minutes to assess cell viability. Fluorescence intensity was measured with a fluorescence plate reader (Fluostar Optima, BMG Labtech, Ortenberg, Germany). Cell viability was compared with that of vehicle control. Cytotoxicity was also measured with the level of LDH released from the damaged cells into the medium culture with the available commercial LDH activity assay kit (Sigma‐Aldrich, Cat. No. MAK066 Sigma‐Aldrich, Budapest, Hungary) according to the manufacturer's instructions.
Calcein acetoxymethyl ester
Manufactured by Merck Group
Sourced in United States, Germany
Calcein acetoxymethyl ester is a fluorescent dye used in various scientific applications. It is a cell-permeant dye that can be used to monitor intracellular calcium levels. The dye enters cells and is hydrolyzed by intracellular esterases, releasing a fluorescent compound that is retained within the cell.
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Lab products found in correlation
Fluostar optima, by BMG Labtech (1 mentions)
Mak066, by Merck Group (1 mentions)
Fluorescence plate reader, by Agilent Technologies (1 mentions)
Ethidium homodimer, by Merck Group (1 mentions)
Ficoll plaque plus, by GE Healthcare (1 mentions)
Serum free endothelial cell basal medium, by PromoCell (1 mentions)
Lsrfortessa flow cytometer, by FlowJo (1 mentions)
Accuri c6, by BD (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Model bh2 rfl t3, by Olympus (1 mentions)
Axiovert 200m fluorescence microscope, by Zeiss (1 mentions)
Image it live mitochondrial transition pore assay kit, by Thermo Fisher Scientific (1 mentions)
Metamorph, by Molecular Devices (1 mentions)
Terephthaloyl chloride, by Merck Group (1 mentions)
Basal endothelial medium, by PromoCell (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Huvecs, by Merck Group (1 mentions)
Triethylamine, by Thermo Fisher Scientific (1 mentions)
1 6 hexanediol, by Merck Group (1 mentions)
1 2 dichlorobenzene, by Thermo Fisher Scientific (1 mentions)
10 protocols using calcein acetoxymethyl ester
1
Calcein-AM Cell Viability Assay
2
Quantifying Cellular Labile Iron Pool
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The total cellular LIP was detected based on the calcein-acetoxymethyl ester method. Cells were treated with 2 μM calcein acetoxymethyl ester (Sigma-Aldrich) at 37 °C for 30 min, then washed with hanks balanced salt solution. The final concentration of 100 μM deferoxamine mesylate is used to remove the iron in calcein. Then, the cells were incubated with or without deferoxamine for 1 h at 37 °C. Fluorescence was measured at 485 nm excitation and 535 nm emissions using the fluorescence plate reader (BioTek, Winooski, VT, USA). The fluorescence change was used as an indirect measurement of LIP after the addition of deferoxamine. The Fe2+ level in cells or mitochondria was detected using an iron detection kit (Sigma-Aldrich). The tissues or the cells collected were immediately homogenized with phosphate-buffered saline (PBS). After centrifugation, the supernatant was assayed for iron concentration using the Kit according to the manufacturer’s instructions.
3
Chondrocyte Viability Assessment in 3D Beads
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Viable and dead cells were distinguished in the slices of TGF-β1-transduced chondrocyte-atelocollagen mixture beads using fluorescent dyes. The gel beads were sliced into 1-mm thick sections to which 2 μM calcein acetoxymethyl ester and 4 μM ethidium homodimer (Sigma-Aldrich) were added. The beads were observed under a fluorescence microscope after 30 min.
4
PBMC Adhesion Assay on HCAECs
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PBMC adhesion assay was performed as previously described [20 (link)]. Briefly, fresh human PBMCs obtained from four different healthy blood donors were isolated by density gradient centrifugation (Ficollplaque plus, 17-1440-02, GE Healthcare Europe). PBMCs were counted and labeled for 30 min with fluorescent tracer calcein acetoxymethyl ester (10 μmol L−1) (Sigma-Aldrich). Labeled PBMCs were added for 1 h at 37 °C to previously transduced HCAECs, cultured for 24 h in serum-free endothelial cell basal medium (Promocell). After washing with PBS, adherent PBMCs were counted in five different fields (Leica TCS SP microscope, 40× magnification). Results were normalized to HCAECs protein level for each condition.
5
Quantification of Intracellular Labile Iron
6
Quantifying Cellular Iron Chelation
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HT29, DLD-1, and Caco-2/BBe cells were incubated for 15 min at 37°C and 5% CO2 with 0.5 μM calcein acetoxymethyl ester (Sigma) to allow for formation of calcein-LIP complex in the cytosol. The cells were washed twice and treated with either Ent (25 µM) or Ent+ Fe3+ (at equimolar ratio) for 3 h. In principle, the iron-chelating property of Ent would compete for the LIP, thus releasing calcein whose fluorescence can be quantified based on the change in mean fluorescence intensity compared with control (ΔF).32 (link),33 (link) Following washing with PBS, cells were analyzed by using Accuri c6 flow cytometer (BD Biosciences, San Jose, CA): cells were gated on forward/side scatter plot and presented as cell count against calcein positivity detected on FL1 channel. The MFI was determined using the Accuri c6 Software (BD Biosciences, San Jose, CA). The magnitude of iron chelation (LIP, ΔF) was calculated by subtracting the difference in the MFI, before and after treatment, with Ent as previously described.16 (link),68 (link),69 (link)
7
Measuring Labile Iron Pool in Hepatocytes
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LIP was measured as described previously. In brief, primary hepatocytes were incubated with 0.05 μM calcein acetoxymethyl ester (Sigma-Aldrich, 17783) for 30 min at 37 °C. The cells were then washed with PBS and incubated with deferiprone (DFO, 100 μM) for 1 h at 37 °C or left untreated. The cells were analysed using a flow cytometer. The difference in the mean cellular fluorescence with and without DFO incubation reflected the level of LIP.
8
Quantifying Spheroid Viability and Necrosis
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Spheroids were washed with 100 µL phosphate buffered saline (PBS), and afterwards incubated with propidium iodide (Sigma-Aldrich, St. Louis, USA; 1:500 in medium) for 5 h 45 min followed by the addition of calcein acetoxymethyl ester (Sigma-Aldrich; 4 mM in dimethyl sulfoxide, 1:800 in medium) with further incubation for 15 min. Spheroids were then transferred to flat-bottom 96-well plates, washed with PBS and imaged under an IMT-2 Inverted fluorescence Microscope (Olympus) equipped with a 100 W Mercury Power Supply (Model BH2-RFL-T3, Olympus) for fluorescence detection. Diameters of total spheroid and necrotic core were measured using the Digimizer software version 5.7.2 (MedCalc Software Ltd, Ostend, Belgium).
9
Quantifying Mitochondrial Pore Opening
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Permeability transition pore complex opening was assayed as previously described62 (link). Cells were loaded with 1 mM calcein acetoxymethyl ester (Sigma-Aldrich, Hamburg, Germany) and Co2+ as instructed by the Image-IT® LIVE Mitochondrial Transition Pore Assay Kit (Thermo Fischer Scientific, Schwerte, Germany). Cells were then imaged based on 490 ± 20 nm excitation and 525 nm long pass emission filters with an Axiovert 200 M fluorescence microscope equipped with a 40X water immersion objective (N.A. 1.2, from Carl Zeiss Microscopy, Jena, Germany). Finally, images were analyzed with MetaMorph® (Molecular Devices, LLC, San Jose, CA, USA), and quenching rate was calculated as the slope of the fluorescence trace over a period of 60 sec after stimulation.
10
Isolation and Culture of HDMECs
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Calcein-acetoxymethyl ester (Sigma Aldrich), Chloroform HPLC (Biosolve BV, stabilized with amylene), Chloroform 99% (Chem-Lab, stabilized with amylene), Chloroform-d 99.6% atom%D (TCI Europe N.V.), Cryopreserved HUVEC (Lonza), 1,10-Decanediol 98% (Sigma Aldrich), 1,2-Dichlorobenzene 99% (Acros organics), 1,12-Dodecanediol 99% (Sigma Aldrich), Endothelial basal medium (Promocell), 1,7-Heptanediol >98% (TCI Europe N.V.), 1,6-Hexanediol 99% (Sigma Aldrich), HUVECs (Sigma Aldrich, pooled), Methanol >99.8% (ChemLab), 1,9-Nonadediol 98% (Sigma Aldrich), 1,8-Octanediol 98% (Sigma Aldrich), 1,5-Pentanediol (Fluka), Sodium hydroxide (Sigma Aldrich), SupplementMix (Promocell), Terephthaloyl chloride ≥99% (Sigma Aldrich), Triethylamine (Fischer Scientific), 1,11-Undecanediol 95% (Chemreagents) were used as received.
HDMECs were isolated with approval of the Medical Ethical Committee from cutaneous breast tissue (UZ Ghent, Belgium, EC2017/0710). The tissue was obtained by performing a subcutaneous mastectomy with 3 healthy Caucasian donors of 23, 29 and 31 years old with a mean body mass index of 24, 29, 25 kg/m 2 respectively, while the cells were isolated as described earlier. 26
HDMECs were isolated with approval of the Medical Ethical Committee from cutaneous breast tissue (UZ Ghent, Belgium, EC2017/0710). The tissue was obtained by performing a subcutaneous mastectomy with 3 healthy Caucasian donors of 23, 29 and 31 years old with a mean body mass index of 24, 29, 25 kg/m 2 respectively, while the cells were isolated as described earlier. 26
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