Facs fortessa x20 analyzer

Following cell stimulation, 3 × 10⁵ PBMCs were stained for 30 min, 4°C, in the dark with the following anti-human antibodies for surface antigen detection: CD45-BUV395 (HI30), CD3-BUV737 [SK7 (also known as Leu-4)], CD4-APC (SK3 (also known as Leu3a)), CD8-BV605 (SK1), CD56-BB700 (NCAM16.2), and CD19-BV650 (SJ25C1), all purchased from BD Biosciences (BD, San Jose, CA, USA) according to the manufacturer’s information. For intracellular cytokine detection, cells were fixed and permeabilized using the Cytofix/Cytoperm Fixation and Permeabilization Kit (BD) for 10 min at 4°C. Cells were then washed in 1× Perm/Wash Buffer (BD) and stained for 30 min, 4°C, in the dark, with IFNγ-BV480 (B27) and IL4-PE (MP4-25D2). Following doublet exclusion and morphological FSC-A/SSC-A setting, total leukocytes were gated on CD45⁺ cells. Samples were acquired using a BD FACS Fortessa X20 analyzer, equipped with 5 lasers. Cell subsets were identified as CD45⁺ cells: CD3⁺ cells (total T cells), CD3⁺CD4⁺ cells (CD4⁺T cells), CD3⁺CD8⁺ cells (CD8⁺ T cells), CD3^-CD19⁺ cell (B cells), and CD3^-CD56⁺ (total NK cells). Total NK cells were interrogated for CD56 subsets (CD56^bright, CD56^dim). Finally, the production of IFNγ and IL4 was interrogated for CD4⁺ and CD8⁺ T cells, B cells, and total NK cells. Flow data were analyzed using the FlowJo v10 software (Tree Star).

For flow cytometry, PC3 cells stably expressing WT or T592E SAMHD1 were plated at 5 × 10⁵ cells per dish in 100 mm dishes, and allowed to grow for 24 h before switching to serum-free media for 36 h. Cells were treated or not with LPA and collected at each time point by trypsinizing cells off the plate. Cells were centrifuged, resuspended in PBS with 1% FBS to wash, and centrifuged again to collect. Samples were resuspended in 500 µL of PBS with 1%FBS, added dropwise to ice-cold ethanol while slowly vortexing, and stored at −20°C. To analyze, cells were centrifuged, washed in ice-cold PBS with 1% FBS, and resuspended in flow solution (100 mM NaCl, 3.6 mM trisodium citrate, 0.6% NP-40, 0.05 mg/ml propidium iodide, 0.1 mg/ml RNAse). Samples were incubated in the dark at 37°C until analysis on a BD FACSFortessa X-20 analyzer. Results were analyzed by FloJo software using the Dean-Jett-Fox model. The mean of n = 14 WT samples and n = 15 T592E samples was calculated. Statistical analysis was performed by two-way ANOVA and intergroup comparison by Tukey-Kramer post-hoc test using RStudio (R Version 4.0.2, RStudio Version 1.1.453) (RStudio Team, 2020 ).