Pharmingen pe annexin 5 apoptosis detection kit 1

To quantify cell apoptosis, Annexin V and 7‐AAD were measured using BD Pharmingen PE Annexin V Apoptosis Detection Kit I (BD Biosciences). MitoTracker Green FM (MG) and MitoTracker Orange CM‐H₂TMRos (MO) (Invitrogen) were used to detect mitochondrial mass and oxidation, respectively. CellROX Green reagent (ThermoFisher) was used to measure cellular ROS. All the assays were performed according to the manufacturers' instructions. The stained cells were analyzed by an Accuri C6 flow cytometer (BD), and the data were analyzed by FlowJo software (Tree Star). Unstained cells were used to determine the background levels of staining and adjust multicolor compensation as a gating strategy.

For the cellular death assay, the BD Pharmingen PE Annexin V Apoptosis Detection Kit I (BD Biosciences) was used. Each sample (n = 5) was washed in Binding Buffer 1x and centrifuged at 400 g during 5 minutes. After centrifugation, the supernatant was removed and the cells were re-suspended in 100 μL of Binding Buffer. Phosphatidylserine, which is a marker of early apoptosis, was stained with PE-(phycoerythrin) labelled Annexin V. Loss of membrane integrity as a consequence of necrosis and late apoptosis was detected by 7-aminoactinomycin D (7-AAD) staining of DNA. Data acquisition was performed with a FACS Calibur flow cytometer (BD Biosciences Immunocytometry Systems).

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)).

Garcinol (sc-200891A, HPLC purity ≥95%) and Z-VAD-FMK (sc-3067, HPLC purity ≥95%) purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 20 mM stock and stored at −20 °C until use. For different assays, the stock was further diluted using cell growth medium as appropriate. Dimethyl sulfoxide (DMSO), served as vehicle and negative control. BD Pharmingen™ PE Annexin V apoptosis detection kit I (#559763) was purchased from BD Biosciences (San Jose, CA, USA). Unless otherwise indicated, all reagents were obtained from Gibco (Thermo Fisher Scientific, Life Technologies, Foster City, CA, USA).