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15 protocols using cyan adp analyzer

1

Flow Cytometry Analysis of Calcium Flux

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Flow cytometry was usually performed using a CyAn ADP analyzer (Dako). Some experiments were performed using an LSR Fortessa or a FACSCalibur analyzer (BD). Calcium fluxes were detected by flow cytometry, as previously described (Dong et al., 2009 (link)). Briefly, Indo-1-loaded thymocytes were stimulated by different pore-forming agents, and fluorescence was monitored over time using an LSR Fortessa analyzer. Calcium flux was quantified as the ratio of emission at 400 nm (bound Ca2+) versus emission at 475 nm (free Ca2+). This ratio is expressed in arbitrary units (AU).
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2

Quantitation of Hyaluronan Binding

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For quantitation of HA binding, cells (HDLEC- or hLYVE-1 pHR-transfected Jurkat) were resuspended in FACS buffer (5% FCS, 5 mm EDTA, 0.05% NaN3 in PBS) and incubated with biotinylated HA (5 μg/ml) either alone or in combination with LYVE-1 mAbs (5 μg/ml), isotype-matched control Ig (5 μg/ml), or excess unlabeled HA (200 μg/ml) for 30 min at room temperature. Cells were then washed (three times) and reincubated in buffer containing appropriate Alexa 488-conjugated secondary antibody (10 μg/ml) and streptavidin Alexa 647 (5 μg/ml) for 30 min at 4 °C, followed by fixation in 2% (w/v) formaldehyde, 0.02% NaN3 in PBS and analysis by flow cytometry (Cyan ADP Analyzer, Dako). The numbers of events analyzed in FACS plots (see Figs. 13) were normalized using proprietary Summit version 4.3 software. In some experiments, the concentration of LYVE-1 mAb was varied (0–800 nm) to determine the relationship between the extent of receptor cross-linking and bHA binding. In others, the cells were incubated with bHA either before or after cross-linking with various LYVE-1 mAbs, with three washes between incubations, to establish the importance of sequence order. All other parameters were maintained as described.
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Quantifying P-glycoprotein Expression

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For Pgp immunodetection, MP derived from Lucena cells, MP derived from parental K562 cells, and recipient cell lines after 24 h of co-culturing (MCF7 and A549) were blocked with 1% BSA for 15 min. Pgp cell surface expression was measured after incubation with an anti-Pgp PE-conjugated monoclonal antibody (clone UIC2; Coulter, Brea, CA, USA) for 30 min through flow cytometry according to the manufacturer's instructions. (FACScalibur, BD or CyAn ADP Analyzer, Dako, Fort Collins, CO, USA).
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4

Measuring Cell Death by Flow Cytometry

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To assess cell death by flow cytometry, cells were first incubated with NaAsO2 and N65828 for the indicated time points or UV light irradiated and left them recovered for the indicated time points. Cells were then shortly trypsinised and disaggregated by gentle pipetting. Trypsin was inactivated by adding media containing FCS. The cells were pelleted and re-suspended in binding buffer and stained for Annexin V (Annexin V Apoptosis detection kit, eBioscience) according to manufacturer's recommendations. Flow cytometry analysis was carried out on a CyAn ADP analyzer (Dako Cytomation), and data were processed in FlowJo.
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5

Cell Cycle Analysis and Clonogenic Survival

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For cell-cycle analysis, cells were treated with 10 μM bromodeoxyuridine (BrdU, Becton Dickinson, 51-2420KC) for 30 min, fixed with 70% Ethanol and stained with FITC-conjugated anti-BrdU antibody (BD Pharmingen 556028), propidium iodine (PI, Sigma Aldrich P4170) and RNase A (Sigma Aldrich 4642). BrdU and propidium iodide staining were evaluated using a CyAn ADP Analyzer (Dako). To examine cell-cycle phase post-transfection, cells were transfected as for the reporter assays, but scaled 2-fold and including 200 ng pgk-puro plasmid. These cells were treated the next day with puromycin (3 μg/ml) for 1 day to enrich for transfected cells prior to BrdU labeling and fixation. To examine clonogenic survival after IR treatment, cells were treated with either 0, 1.5 or 3 Gy of IR (Gammacell 3000), and seeded at low density to form colonies, which were fixed (10% acetic acid, 10% methanol), stained with 1% crystal violet and counted under the microscope with a 10× objective. To calculate the frequency of clonogenic survival, the number of colonies per well were normalized to the number of cells plated and this colony forming value for each treatment was divided by the mean value of the parallel untreated plates (0 Gy).
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6

Quantitative Binding Assay for LYVE-1

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To prepare multimers, bHA (5 μg/ml) was initially added to streptavidin Alexa 647 (SA647) over a range of concentrations from 0.002 to 20 μg/ml for 30 min at room temperature to determine the optimal bHA/SA647 ratio for LYVE-1 binding. Subsequently, complexes were prepared using a ratio of 2:1 (w/w) by mixing bHA (10 μg/ml) and SA647 (5 μg/ml). For binding studies, hLYVE-1 pHR-transfected Jurkat cells or HDLEC were resuspended in FACS buffer (PBS, pH 7.5, supplemented with 5% (v/v) FCS, 5 mm EDTA, and 0.05% w/v NaN3) and incubated with bHA·streptavidin Alexa 647 multimers or control uncomplexed bHA (10 μg/ml) either alone or in combination with LYVE-1 HA-blocking mAb 891 (20 μg/ml) or an excess of unlabeled HA (200 μg/ml) for 40 min at 4 °C. After washing (three times), cells were fixed, and the amount of bound HA·SA647 multimer was quantitated by flow cytometry using a Cyan ADP Analyzer (Dako).
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7

Redox Sensing Probes Oxidation Analysis

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Oxidation of the roGFP and mito-roGFP probes was measured by flow cytometry. Briefly, cells were infected with adenoviral vectors expressing roGFP and mito-roGFP sensors at 100 PFU per cell and incubated for 6 hrs after which time the virus-containing media was replaced with complete RPMI 1640 media and incubated overnight. Cells were harvested and equal aliquots were transferred to tubes with media, media with 1mM dithiothreitol or media with 1mM t-butylhydroperoxide (t-BOOH). After incubation for 10 mins at room temperature, the ratio of fluorescence emission at 535 nm and excitations at 405 and 488 nm was measured in 5,000 cells/sets using a Dako Cytomation CyAn ADP analyzer. The roGFP ratio was obtained by dividing the emission values obtained at 488 nm/535nm by that obtained by 405 excitation/535nm emissions. The oxidation state of the cells was calculated as the difference in the ratio observed with untreated values and completely reduced values divided by the difference in the ratio observed with DTT and t-BOOH. Values are represented as the percentage of oxidation of the probe with 100% being fully reduced and 1% being fully oxidized (18 (link)).
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8

Splenocyte Phenotyping by Flow Cytometry

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Splenocytes isolated from each mouse were stained with antibodies to surface or intracellular antigens and incubated for 30 min at room temperature. Labeled cells were washed and suspended in 150 µL of PBS and evaluated by flow cytometry, as previously described [14 (link)]. In brief, intracellular antigens were stained by incubating 500 µL suspensions of the cell pellets with fixation/permeabilization buffer (eBioscience, San Diego, CA, USA) in the dark for 45 min. Cells were then treated with permeabilization buffer (eBioscience, San Diego, CA, USA), and incubated with an intracellular antibody mix for an additional 30 min at 4 °C. All antibodies were obtained from eBiosciences and Biolegend (San Diego, CA, USA) (Supplemental Table S1). Supplemental Table S2 lists the immune cell types analyzed as well as corresponding fluorochrome-labeled antibodies used for analysis. Flow cytometry was performed with a CyAn ADP Analyzer (Dako, Ft Collins, CO, USA). Gatelogic software (eBioscience) was used for data analysis.
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9

Analyzing Cell Cycle and Genome Editing

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To examine cell cycle phase, cells were incubated with 10 μM bromodeoxyuridine (BrdU) for 30 minutes, fixed with 70% Ethanol, and stained with mouse monoclonal anti-BrdU FITC antibody, propidium iodine, and RNase A. Staining was evaluated using a CyAn ADP Analyzer (Dako). Mutagenic end joining frequencies were determined for cells transfected as for the reporter experiments, using the Cas9/sgRNA plasmid targeting the 19 kbp DSB/repeat distance. Genomic DNA was isolated and examined with the Surveyor Mutation Detection Kit, as described, using the primers Surveyor P1 and P2 (Table S1) (Mendez-Dorantes et al., 2018 (link)). Predictions for G-quadruplex forming repeats was performed with the non-B DNA Motif Search Tool (Cer et al., 2013 (link)).
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10

Isolation and Flow Cytometry Analysis of Mouse Splenocytes

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Mouse splenocytes were isolated as previously described [12 (link)] with a final concentration of 5×106 cells/mL. S1 Table lists the immune cell types analyzed and corresponding fluorochrome-labeled antibodies. Cells were mixed with permeabilization buffer (eBioscience, San Diego, CA, USA), centrifuged, resuspended and analyzed by flow cytometry as previously described [12 (link)] using a CyAn ADP Analyzer (Dako, Ft Collins, CO, USA). Flow data was analyzed using Gatelogic software (eBioscience).
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