Fi 1000

HEK293T cells were grown on 0,01% poly-L-lysine coated coverslips and analyzed 24 hours post-transfection with DNAs coding: IFITMs, HIV-1 Gag-Pol plus non-structural viral proteins, NL4-3 Env, a miniviral genome coding CD8, as well as a small amount of Gag-GFP (ratio 3:1:1:1:0,1). After Formalin fixation, cells were incubated with the following antibodies: anti-Flag (F7425, Sigma), followed by DyLight 649- conjugated sheep, or FITC-conjugated goat anti-rabbit IgG (STAR36D649 AbD serotec and FI-1000 Vector). DAPI-containing mounting medium was finally used (DAPI Fluormount G, Southern biotech). Images were acquired using a spectral Leica sp5 and analyzed with the Fiji software [54 (link)]. Two-dimensional graphs representing pixel intensities (gray level) were plotted along a 30-μm lines (yellow on Figure 3B), using Plot Profile tool in Image-J. The extent of reciprocal co-localization between Gag and IFITMs was quantified using the Manders overlap coefficient (Fuji image software) on more than 40 cells per condition.

Mouse N2a neuroblastoma cells were subjected to OGD for 4 hours followed by reperfusion for 0, 4, and 12 hours. The cells were then fixed with 4% paraformaldehyde for 30 min and washed three times with PBS, pH 7.4. The cells were incubated with a primary rabbit anti-Tom20 antibody (1 : 200; SC-11415, Santa Cruz Biotechnology) overnight at 4°C. On the following day, the cells were incubated with fluorescein-conjugated anti-rabbit IgG (1 : 400; FI-1000, Vector Laboratories) for 1 h. N2a cells were counterstained with 1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA) to visualize nuclear morphology. Slides were washed, wet-mounted, and observed using a confocal microscope (Zeiss LSM Meta710, 63x/1.4 objective). Mitochondria fragmentation was assessed visually and quantified as percentage of control. Cells that displayed a network of filamentous mitochondria were classified as normal. Cells with fragmented or partially reelongated mitochondria were classified as fragmented [7 (link)].

Cell cultures from passage 2 skMPCs were analyzed by fluorescence-activated cell-sorting analysis (FACS analysis), 200,000 cells of each of the fixed samples from the monkeys were immunolabeled. All cell samples were pelleted and blocked with the use of Fc Blocking Reagent (Miltenyi Biotec Inc., Auburn, CA, USA) for 15 minutes. Immunolabeling followed immediately with mouse monoclonal anti-CD34 (550619, BD Pharmingen, San Diego, CA, USA) antibody, mouse monoclonal anti-CD44 (550989, BD Pharmingen) antibody, mouse monoclonal anti-CD45 (558411, BD Pharmingen), skeletal muscle actin (1:100, Thermo Fisher Scientific, Waltham, MA, USA, PA1-37019), MyoD (myoblast determination protein) (1:100, Pharmingen), or CD117 (1:100, BioLegend, San Diego, CA, USA, 104D2). After incubating for 1 hour at 4 °C all tubes were washed three times and labeled using species-specific FITC (FI-1000, Vector Laboratories, Burlingame, CA, USA). A negative control (no primary antibody, species-matched serum) was run simultaneously with each experimental sample. Cell fluorescence was measured immediately after staining with a Becton Dickinson FACS Calibur flow cytometer (BD Biosciences, San Diego, CA, USA) and data analyzed using FlowJo software v. 7.1.3 (Tree Star Inc., Ashland, OR, USA).