Guava incyte software

IgLON5_wt and IgLON5_kd cells were cultured in growth medium for 3 days, trypsinized, washed twice with ice-cold PBS, and left overnight in 70% ethanol at −20 °C. The ethanol was then removed by centrifugation, and cells were washed twice with ice-cold PBS. Cells were then treated with cell cycle reagent (Merck Millipore, Darmstadt, Germany) for 30 min in the dark at room temperature and performed with flow cytometry (EasyCyte5HT; Merck Millipore, MA, USA). The measurement data were analyzed by Guava^® InCyte software (Merck Millipore, MA, USA).

To evaluate cell susceptibility to extrinsic stimuli, XFM and SCM cells (5 × 10³ cells/cm²) were seeded into 12-multiwell plates and treated with 1 μM staurosporine (Wako Pure Chemical Industries) or 100 μM hydrogen peroxide (H₂O₂; Wako Pure Chemical Industries) for 6 h at 37 °C, or were exposed to ultraviolet (UV) radiation using a UV transilluminator (UVP, Upland, CA, USA) for 30 min at room temperature. staurosporine, which is a known chemical inducer of apoptosis, was used as a positive control for cell-damage induction. Cell damage was evaluated by flow cytometry using an Annexin V/propidium iodide (PI) system described previously [28 (link)], with minor modifications. Briefly, the treated cells were stained with 100 μl of Guava Nexin® reagent (Merck KGaA) for 20 min at room temperature in the dark and subjected to flow cytometry in the Guava™ EasyCyte HT system equipped with Guava™ InCyte software (Merck KGaA). The data were analyzed with FlowJo software (TreeStar). Annexin V-positivity and PI-negativity indicated the fraction of early apoptotic cells and Annexin V/PI-double positivity indicated the fraction of late apoptotic cells. We considered that both fractions represented damaged cells [28 (link)]. The results were expressed as the ratio of the number of damaged cells to the number of nondamaged cells.

MDCK II Tet-Off cell lines stably expressing Flag-TMIGD1 constructs were washed in PBS, incubated for 10 min at 37 °C in PBS / 5 mM EDTA followed by incubation with Accutase® (Sigma Aldrich #A6964) for 10 min at 37 °C. For the analysis of cell surface-localized proteins, cells were incubated with rabbit pAb anti-Flag in CellWASH (BD #349524) containing 5% BSA (1 h, 4 °C), followed by incubation with AlexaF488-conjugated anti-rabbit pAb (45 min, 4 °C). For intracellular stainings, cells were washed, fixed in PBS / 2% PFA, and incubated with primary and secondary antibodies in the presence of 0.5% saponin (Sigma Aldrich #A47036). After washing, cells were resuspended in CellWASH containing 5% BSA at 10⁶ cells per ml and analyzed by flow cytometry using a Guava® easyCyte™ flow cytometer (MerckMillipore). For each sample, 10.000 cells were measured. Results were analysed using the Guava® InCyte™ software (MerckMillipore) and are plotted as histograms.

For measuring the TNHs, ZnO NCs or EVs internalization in KB cells, 3 × 10⁴ cells were cultured into a 24-well plate (Corning, 24-well TC-treated microplates) with 500 μl of complete cell culture medium for 24 h. Then, cell medium was replaced with freshly prepared solutions containing 5, 15, 25 μg/ml of TNHs (labeled with both DiO for EVs and Atto647 for the ZnO NCs) or, as control samples, with 5, 15, 25 μg/ml of ZnO NCs (labeled with Atto647) or with DiO-labeled EVs at the same concentration used for the preparation of 15 μg/ml of TNHs. Untreated KB cells were also prepared as reference. After 24 h, cells were washed with PBS, trypsinized, centrifuged at 130 × g for 5 min, and then resuspended in 500-μl PBS for the cytofluorimetric analysis. 1 × 10⁴ events were collected with a Guava Easycyte 6-2 L flow cytometer (Merck Millipore, MA, USA), with 0.59 μl/s flow rate, excluding cell debris. The analyses were performed by using the blue laser (λ_ex = 488 nm) to detect EVs and the red one (λ_ex = 642 nm) for ZnO NCs.
The positive events were characterized by a shift of Red-R fluorescence intensity (emission filter 661/15) for Atto647-ZnO NCs signal and a shift of Green-B fluorescence intensity (emission filter 525/30) for DiO-EVs signal. The percentages of positive events were compared with untreated cells, evaluated with Guava InCyte Software (Merck Millipore).

MCF-7 and MDA-MB-231 cells were seeded in 6-well plates at a density of 8 × 10⁴ cells per well and incubated for 24 h prior to treatment with 0.5 mM or 1 mM PBN. Cells were harvested at 24, 48, and 72 h following treatment, washed twice with phosphate-buffered saline (PBS), centrifuged at 200× g for 5 m at 4 °C, re-suspended in 1 mL of old PBS and fixed in 4 mL of cold absolute ethanol. Prior to flow cytometry analysis, cells were pelleted and treated with 100 µL of 200 µg/mL DNase-free RNase A for an hour at room temperature, and then incubated with 20 μg/mL propidium iodide ([PI]; Sigma) for 15 m in the dark. Cell cycle analysis was performed using a Guava Easy Cyte 8™ flow cytometer operated by Guava InCyte™ software (EMD Millipore, Darmstadt, Germany).

Flow cytometry was performed to characterise A-MSCs. The following cell surface epitopes were detected using anti-human CD29 (eBioscience, 4303570, 1 : 20), CD31 (eBioscience, 4303600, 1 : 20), CD44 (eBioscience, 4330029, 1 : 20), CD45 (eBioscience, 4343394, 1 : 20), CD73 (eBioscience, 4344363, 1 : 20), CD90 (eBioscience, 4307303, 1 : 20), and CD105 (eBioscience, 1983608, 1 : 20). Briefly, 1 × 10⁶ cells were stained with 0.125 μg of each specific fluorescence-labelled antibody in 100 μL of phosphate-buffered saline (PBS) for 20 min at 25°C. Flow cytometry was performed with the Guava easyCyte 6HT (EMD Millipore, Billerica, MA, USA), and the data were examined using the Guava InCyte software (3.1 version, EMD Millipore).