Cellrox green ros detection kit

Cellular ROS in CD4 T cells was detected using the CellROX Green ROS Detection kit according to the manufacturer’s protocol (Thermo Fisher Scientific; Waltham, MA, USA). Mitochondrial ROS was assessed by staining cells with 5 μM MitoSOX Red mitochondrial superoxide indicator (Invitrogen; Waltham, MA, USA) at 37 °C for 30 min, washed 3 times, and then analyzed using flow cytometry. For mitochondrial function analysis, the MG (Cat #M-7514) and MO (Cat #M-7511) (Invitrogen) probes were used according to the manufacturer’s instructions. The MG and MO mitochondrion-selective probes allow for the quantification of mitochondrial mass and oxidative phosphorylation (OXPHOS), respectively. The cells were cultured with 100 nM of MG or 500 nM of MO for 30 min at 37 °C. For apoptosis analysis, the cells were washed twice with DPBS and stained with Annexin V-PE (Cat #BDB556422) (BD Biosciences; San Jose, CA, USA) in 1X binding buffer for 15 min according to the manufacturer’s protocol. Controls for these assays included unstained cells, isotype control antibodies, and single positive staining, which were used for gating and compensation. Samples were analyzed using a BD AccuriC6 Plus flow cytometer and FlowJo V10 software.

For phenotypic analysis of CD4 T cells, PBMCs were stained with CD4-FITC, CD8-PE, CD57-APC (BioLegend, San Diego, CA), CD28-PE (Invitrogen, Carlsbad, CA), CD45RA-PerCP710, PD1-FITC (eBioscience, San Diego, CA), or isotype control antibodies. To quantify cell apoptosis, PBMCs were stained with CD4-A647 and CD45RA-FITC for naïve or memory cell populations, and then stained with Annexin V-PE and 7-AAD using BD Pharmingen™ PE Annexin V Apoptosis Detection Kit I (BD Biosciences, San Jose, CA). CD4⁺ T cells were also stained for caspase-3 expression following a protocol from CaspGLOW™ Fluorescein Active Caspase-3 Staining Kit (Invitrogen). Levels of reactive oxygen species (ROS) in CD4 T cells were measured using the DCFDA-based Cellular ROS Detection Kit (Abcam, Cambridge, MA) or CellROX Green ROS Detection kit (ThermoFisher Scientific, Waltham, MA) according to manufacturer's protocol. For intracellular staining, the cells were fixed and permeabilized with Foxp3 Transcription Factor Staining Buffer Set (eBioscience), and stained with pATM (Ser1981)-PE antibody (BioLegend), pCHK2 (Thr68)-PE antibody, γH₂AX-PE (eBioscience), IL-2-PE, and IFN-γ-PE (Invitrogen). Flow cytometry was carried out as described previously (19 (link), 20 (link)).