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28 protocols using Calcein-AM

After 24 h reoxygenation, cell viability was tested with the calcein AM assay. Cells were washed with PBS (pH 7.0) and incubated with calcein AM (1μM; Anaspec, Fremont, CA, USA) in PBS for 15 min at 37 °C. Fluorescence was measured using a Tecan Infinite F200 plate reader (Maennedorf, Switzerland) with 485/530 nm excitation/emission. Percent viability was calculated by comparing to the corresponding control. Treatment groups consisted of four independent trials, each in sextuplicate. After spectrophotometric analysis, green fluorescent images were obtained using a Zeiss Observer Z1 fluorescence microscope.
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SKOV-3 or MEF cell lines were pretreated with 10 μm LPA (Avanti) for 1 h in 0.1% fatty acid-free BSA-containing medium, followed by incubation with 50 μm cisplatin for 20 h (SKOV-3) or 2 μm adriamycin or 8 h (MEFs). Caspase-3/7 activity was determined by cleavage of the luminogenic substrate containing the DEVD sequence (Promega) and was normalized to protein concentrations. Alternatively, cell viability was determined by calcein/propidium iodide double fluorescence staining of the live/dead cells. After treatment was complete, cells were incubated with 0.5 μm calcein-AM (AnaSpec, Fremont, CA, USA) and 0.1 μg ml−1 propidium iodide (Roche, Indianapolis, IN, USA) for 10 min at room temperature. Images of green fluorescent calcein-positive live cells and red fluorescent propidium iodide-positive dead cells were acquired under fluorescence microscope using ×20 long-ranged objectives. Totally 500–1000 cells per sample were counted to determine the percentage of live and dead cells.
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Acini were loaded by incubation with 1uM Calcein/AM (ANASPEC) dissolved in solution A for 10 min at room temperature. Acini were continuously perfused and fluorescence was recorded every 2 seconds at excitation wavelengths of 490 nm, and emitted light at wavelength higher than 510 nm was collected. Images were analyzed with MetaFluor software. At the end of each experiment the signals were calibrated by exposing the cells to a solution of 20% reduced osmolarity and the signal was taken as 20% change in cell volume.
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SKOV-3 or MEF cell lines were pretreated with 10 μM LPA (Avanti) for 1 h in 0.1% fatty acid-free BSA-containing medium, followed by incubation with 50 μM cisplatin for 20 h (SKOV-3) or 2 μM adriamycin or 8 h (MEFs). Caspase-3/7 activity was determined by cleavage of the luminogenic substrate containing the DEVD sequence (Promega) and was normalized to protein concentrations. Alternatively, cell viability was determined by calcein/propidium iodide double fluorescence staining of the live/dead cells. After treatment was complete, cells were incubated with 0.5 μM calcein-AM (AnaSpec, Fremont, CA, USA) and 0.1 μg ml−1 propidium iodide (Roche, Indianapolis, IN, USA) for 10 min at room temperature. Images of green fluorescent calcein-positive live cells and red fluorescent propidium iodide-positive dead cells were acquired under fluorescence microscope using × 20 long-ranged objectives. Totally 500–1000 cells per sample were counted to determine the percentage of live and dead cells.
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Control and pretreated MSCs were trypsinized and labeled with a transmembrane fluorescent viability marker, Calcein AM (Anaspec), in HBSS containing divalent ions Ca++ and Mg++. Then the cells could adhere for 3 h after stimulus was removed (t0 + 3 h) in HBSS before taking an initial florescence reading in a DTX-800 Multimode Detector plate reader. A final reading was taken after removing non-adherent cells by washing with HBSS to determine the adherent fraction.
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For migration and adhesion experiments, isolated neutrophils (1 × 107/ml in HBSS) were incubated with the fluorescent dye calcein am (Anaspec, Fremont, CA) at 2 ug/ml for 30 minutes at room temperature. Cells were then centrifuged at 1000 rpm for 8 min and resuspended in chemotaxis buffer to the appropriate final experimental concentration.
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The cell viability of Coleman fat and ACF was evaluated using live/dead staining. Briefly, 1 ml of PBS supplemented with 2 μl of Calcein-AM (1 mg/ml) (AnaSpec, Fremont, Calif.) and 2 μl of PI (1 mg/ml) (Sigma-Aldrich, St. Louis, MO, United States) was added to 1 ml of Coleman fat or ACF samples. After incubating for 10 min at 37°C, the samples were observed under a fluorescence microscope (BX 51, Olympus, Japan). The experiment was independently repeated three times.
The explant culture was performed according to a standard protocol (Sineh et al., 2020 (link)). Briefly, the Coleman fat and ACF were evenly distributed on the surface of 100 mm culture dishes (1 ml/dish). The samples were cultured in complete growth medium (HUXMD-90011, Cyagen, China) at 37°C with 5% humidified CO2 for 5 days. Then, the outgrown cells from explants were observed under a light microscope (GX53, Olympus, Japan). The experiment was independently repeated three times.
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The 300-PEO-PEOT/PBT 55/45 was acquired from PolyVation™ (Groningen, The Netherlands) in the form of pellets and processed without prior purification. Chloroform 99%, anhydrous (CHCl3) was purchased from Sigma Aldrich (St. Louis, MO, USA). Glass cover slips (Ø = 12 and 25 mm) were ordered from VWR (Radnor, PA, USA) and cleaned with ethanol prior to use. Ar, He, N2 and dry air (Alphagaz 1) were ordered from Air Liquide (Paris, France).
Human foreskin fibroblasts (HFF-1) were acquired from ATCC (Manassas, VA, USA). DMEM glutamax, penicillin (10 U/mL), streptomycin (10 mg/mL), fetal bovine serum (FBS) and sodium pyruvate were ordered from Gibco Invitrogen (Waltham, MA, USA). Calcein AM (1 mg/mL) was purchased from Anaspec (Fremont, CA, USA) and propidium iodine was ordered from Sigma Aldrich (St. Louis, MO, USA). Yellow tetrazolium dye 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was acquired from Merck Promega (Darmstadt, Germany).
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To assess how 24 hour MTX treatment (2.5 μM) affects cell viability, ASCs and NHFs were monitored using a live/dead assay. Cells were stained on the day of MTX treatment removal (day 0) as well as the following ten days by incubating cultures in 2 μM calcein AM (AnaSpec Inc, Fremont, CA), 4.5 μM propidium iodide (EMD Millipore), and 1 μg/mL Hoechst for 30 minutes. Cellular viability was quantified by taking 16-24 images/well (N = 3 wells) at 10× and counting stained cells (n > 200).
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The cell experiments consist of testing the cell viability of mouse calvaria 3T3 (MC3T3) sub clone pre-osteoblast cells on untreated and plasma-activated samples. Before culturing the cells, the samples are subjected to UV sterilization for 30 min. The cells are cultured with a density of 20000 cells per ml, with 1 ml per sample. After 24 h and after 7 days, cell viability is analyzed with a CellTiter 96® aqueous non-radioactive cell proliferation assay (Promega, USA). This MTS assay measures the cellular conversion of MTS into a soluble formazan dye by the mitochondrial NADH/NADPH-dependent dehydrogenase. The absorbance of the formazan dye in the solution is measured with a 490 nm Universal microplate reader EL 800 (BioTek Instruments, USA). The cell viability is calculated as a percentage of a control culture and each sample condition is tested in triplicate. Additionally, 7 days after cell seeding, a fluorescent live/dead staining with calcein AM (Anaspec, USA) and propidium iodide (Sigma Aldrich, Belgium) is performed, after which the live (green) and dead (red) cells are visualized with a fluorescence microscope (Olympus IX 81) making use of appropriate filters.
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