Attune nxt flow cytometer

Flow cytometry was performed to detect phosphorylated p38 MAPK in RAW 264.7 cells using an Attune NxT flow cytometer (Thermo Fisher Scientific) [17 (link)]. Briefly, RAW 264.7 macrophages were seeded in 6-well plates (1 × 10⁶ cells/well) and incubated with LPS (0.1 µg/mL) and RF for 30 min. After incubation, cells were harvested and washed with Flow Cytometry Staining Buffer (SB). Prior to antibody staining, cells were fixed with the pre-warmed Fix Buffer I for 10 min. Then, cells were washed with SB and permeabilized with Perm Buffer III on the ice for 30 min. Then, cells were stained with 5 µg/mL of phospho-p38 MAPK Antibody (T180/Y182) (eBioscience 17-9078-42), or 1.2 µg/mL of Mouse IgG2b kappa Isotype Control (eBioscience 12-4732-81), and analyzed on the Attune NxT flow cytometer (Thermo Fisher Scientific) using Attune NxT software (Thermo Fisher Scientific).

The disruption of MtMP was assessed by staining with Rhodamine (Rh)-123, a fluorescent probe that is induced via mitochondrial energization [63 (link),64 (link),65 (link)]. N. glabrataa DSM70614 cells were cultivated and exposed to various concentrations of sphingosine at 37 °C. At various timepoints, the cells were harvested and stained with Rh-123 (25 µM for 10 min) and then washed three times with PBS. The mean intensity values of 10,000 events were measured per sample with an Attune NxT flow cytometer (ThermoFisher Scientific). Treatment with 20 mM sodium azide (NaN₃) was used as a positive control. After staining with Rh-123, cells were washed three times with PBS, and the MFI values of 10,000 events were measured per sample with an Attune NxT flow cytometer (ThermoFisher Scientific).

HeLa cells stably expressing GFP-tagged mutant α-synuclein (EGFP-A53T) were treated with siRNA for 48 to 72 hours before analysis. Cells were then trypsinized and GFP fluorescence was analyzed using an Attune NxT Flow Cytometer (Thermo Fisher Scientific) using the BL1 (488 530/30) detector. Cells were first gated on forward (FSC-A) and side scatter (SSC-A) for P1 and then for singlets (FSC-A/FSC-H) for P2. A total of 20,000 single cells were recorded for each replicate. GFP⁺ gates were set using normal unstained HeLa cells. HeLa cells stably expressing SRAI-LC3B were treated with siRNA for 48 to 96 hours before analysis and with SMER28 where indicated. Cells were trypsinized and analyzed using an Attune NxT Flow Cytometer (Thermo Fisher Scientific) using the VL2 (405 512/25) and BL1 (488 530/30) detectors. The ratio of VL2 to BL1 signals was derived for each cell and the median ratio per condition was used for analysis. The data were analyzed using FlowJo software v10.7.1.

HeLa cells stably expressing GFP-tagged mutant α-synuclein (EGFP-A53T) or Ub-G76V-GFP were treated with various compounds for 24 h. Cells were then trypsinized and GFP fluorescence was analyzed using an Attune NxT Flow Cytometer (ThermoFisher Scientific) using the BL1 (488 530/30) detector. Cells were first gated on forward (FSC-A) and side scatter (SSC-A) for P1 and then for singlets (FSC-A/FSC-H) for P2. 20,000 single cells were recorded for each replicate. GFP + gates were set using normal HeLa cells. HeLa cells stably expressing SRAI-LC3B were treated with various compounds for 24–48 h. Cells were trypsinized and analyzed using an Attune NxT Flow Cytometer (ThermoFisher Scientific) using the VL2 (405 512/25) and BL1 (488 530/30) detectors. The ratio of VL2 to BL1 signals was derived for each cell and the median ratio per condition was used for analysis. The data were analyzed using FlowJo software v10.7.1.

A Thermo Scientific Heraeus B12 incubator (Thermo Fisher Scientific, Waltham, MA, USA), Sanyo orbital incubator (Sanyo, Japan), Sanyo autoclave (Sanyo, Japan), Hitachi U-2910 UV/Vis spectrophotometer (Hitachi, Japan), WTW pH meter (inoLab, Germany), Multiskan EX plate reader (Thermo Fisher Scientific Inc., Vantaa, Finland), benchtop centrifuge (Hettich, Buford, GA, USA), Ultramicrotome Reichert Jung Ultracut E (LabX, Midland, ON, Canada), JEOL-1200EX Transmission electron microscope (TEM), and Attune NxT flow cytometer (Thermo Fisher Scientific Inc., Massachusetts, USA) were used throughout the experiments.
A Thermo Scientific Heraeus B12 incubator (Thermo Fisher Scientific, Waltham, MA, USA), Sanyo orbital incubator (Sanyo, Japan), Sanyo autoclave (Sanyo, Japan), Hitachi U-2910 UV/Vis spectrophotometer (Hitachi, Japan), WTW pH meter (inoLab, Germany), Multiskan EX plate reader (Thermo Fisher Scientific Inc., Vantaa, Finland), benchtop centrifuge (Hettich, USA), Ultramicrotome Reichert Jung Ultracut E (LabX, Canada), JEOL-1200EX Transmission electron microscope (TEM), and Attune NxT flow cytometer (Thermo Fisher Scientific Inc., Massachusetts, USA) were used throughout the experiments.

In this study, 4′,6-diamidino-2-phenylindole (DAPI)-stained cells were used to examine the changes in cell morphology during apoptosis under fluorescence microscopy. The cells were fixed with 4% paraformaldehyde for 30 min at room temperature and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline, then incubated with 30 min DAPI (1 μg/mL). In order to check the percentage of apoptotic nuclei, 200 to 300 cells were scored using a fluorescent microscope. Cell-cycle distribution of the cells was analyzed by propidium iodide staining and FACS analysis (Attune NxT Flow Cytometer, Thermo Fisher Scientific Inc., Waltham, MA, USA).
The fluorescent probes of 10 μM 2′, 7′-dichlorofluorescein diacetate (H2DCFDA, Molecular Probes) were used to measure hydrogen peroxide (H₂O₂), which represented the intracellular accumulation of ROS. The cells with H2DCFDA staining were subjected to a FACS analysis (Attune NxT Flow Cytometer, Thermo Fisher Scientific Inc., Waltham, MA USA). The data of the fluorescent intensity and the number of stained cells were quantified and analyzed and were represented as a percentage of the untreated control group in three independent experiments [24 (link)].