Pchk2 thr68 pe antibody

For phenotypic analysis of naive CD4 T cells, PBMCs were stained with CD4-APC, CD45RA-FITC (BioLegend, San Diego, CA, USA) antibodies, or isotype controls. To quantify cell apoptosis, naive or memory cells were purified, cultured, and collected at indicated days and stained with Av and 7AAD using BD Pharmingen PE Av Apoptosis Detection Kit I (BD Biosciences, San Jose, CA, USA). For intracellular staining, the cells were fixed and permeabilized with Foxp3 Transcription Factor Staining Buffer Set (eBioscience, San Diego, CA, USA), and stained with pATM (Ser1981)-PE antibody(BioLegend), ATM antibody (Abcam), and anti-Rabbit-IgG-Alexa Fluor 488 (Santa Cruz Biotechnology, Dallas, TX, USA), pCHK2 (Thr68)-PE antibody (eBioscience), 8-oxoguaine-FITC probe (OxyDNA Assay Kit, EMD Millipore, Billerica, MA). The stained cells were analyzed on AccuriTM C6 flow cytometer (BD, Franklin Lakes, NJ), and data were analyzed by FlowJo software (Tree Star, Inc., Ashland, OR). Isotype control antibodies (eBioscience) and fluorescence minus one controls were used to determine the background levels of staining and adjust multicolor compensation as gating strategy.

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)).