Secondary antibody conjugated to fitc

U87-CD4-CCR5 cells, HIV_ADA, and HIV-1 p24 antibody were obtained from NIH AIDS Reagent program (Germantown, MD, United States). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, trypsin-EDTA, secondary antibody conjugated to FITC, and prolong gold anti-fade reagent with DAPI were purchased from Thermo Fisher Scientific (Waltham, MA, United States). The in situ cell death detection kit, TUNEL was purchased from Roche (Mannheim, Germany). BAPTA-AM, 18α-glycyrrhetinic acid (AGA), and isoflurane were procured from Sigma–Aldrich (St. Louis, MO, United States). Xestospongin C (XeC) was purchased from Tocris (Bristol, United Kingdom). Cx43 blocking peptide and scrambled (Scr) peptide were obtained from PeproTech (Rocky Hill, NJ, United States).

Intracellular reactive oxygen species (ROS) and superoxide production were measured using 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA) and dihydroethidium, respectively, as described elsewhere [72 (link)]. Oxidative DNA damage as a level of 8-hydroxy-2′-deoxyguanosine (8-OHdG, 8-oxo-dG) was measured using Epigentek EpiQuik 8-OHdG DNA Damage Quantification Direct Kit (Gentaur, Poland) as previously described [52 (link)]. Nuclear protein carbonylation was evaluated using nucleus and DNP co-staining. Protein derivatization was conducted according to [81 (link)]. Fixed and derivatized cells were incubated with the primary antibody anti-DNP (1:200) (Abcam) and the secondary antibody conjugated to FITC (1:1000) (Thermo Fisher Scientific). DNA was visualized using DAPI staining. Digital cell images were captured with an Olympus BX61 fluorescence microscope equipped with a DP72 CCD camera and Olympus CellF software. To analyze the level of nuclear protein carbonylation, ImageJ software http://rsbweb.nih.gov/ij/ was used. Briefly, the integrated fluorescence density (green channel) that is the sum of all pixel values within the marked area of each nucleus analyzed and equivalent to the product of the area and mean gray value was evaluated. The integrated fluorescence density is presented in relative fluorescence units (RFUs).