Cyan adp analyzer

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.

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).

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).

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.

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).