Bcl xl shrna gfp lentiviral particles

Primary hippocampal neurons transduced with GFP control lentiviral particles (copGFP, Santa Cruz Biotechnology) or Bcl-xL shRNA-GFP lentiviral particles (Santa Cruz Biotechnology) were imaged with a Zeiss Axio Vert.A1 microscope (Zeiss, Oberkochen, Germany). Sholl analysis was used to quantify neurite branches as previously described [36 (link),37 (link)]. In brief, fluorescent micrographs of primary hippocampal neurons were opened using the Simple Neurite Tracer plugin for ImageJ (National Institutes of Health, Bethesda, MD, USA), and neurites from the soma and daughter branches from the neurites were selected using the Path Manager function. Saved traces were analyzed using Sholl analysis, and a Sholl profile was created with the number of intersections at the specific radium, and micrographs were converted with pseudo-color (16-color annotation).

Primary rat hippocampal neurons were prepared from rat feti (Sprague-Dawley, day 18 of gestation; Envigo, Indianapolis, IN, USA) as described previously [5 (link),32 (link),33 (link),34 (link)]. Briefly, neurons (0.3 × 10⁶ cells/35 mm plate) were seeded on poly-l-lysin coated plates and grown in a neurobasal medium supplemented with B-27, glutamine, and antibiotics (Thermo Fisher Scientific, Waltham, MA, USA) for 21 days in vitro (DIV). shRNA lentiviral particle transduction: Primary hippocampal neurons were transduced with control shRNA lentiviral particles or Bcl-xL shRNA lentiviral particles (Santa Cruz Biotechnology, Dallas, TX, USA); copGFP control lentiviral particles or Bcl-xL shRNA-GFP lentiviral particles (Santa Cruz Biotechnology) at DIV 7. Glutamate treatment: Glutamate (Sigma-Aldrich, St. Louis, MO, USA) was freshly prepared in sterile PBS (Thermo Fisher Scientific) and added to the cell culture medium (final concentration: 20 μM). The vehicle control group for the Glutamate experiment was treated with isovolumetric sterile PBS. ABT-263 treatment: ABT-263 was prepared in dimethyl sulfoxide (DMSO) and added to the cell culture medium (final concentration 1 μM). The vehicle control group for ABT-263 was treated with isovolumetric DMSO. The protocol was approved by the Institutional Animal Care Committee (IACUC) of the University of Alabama, Tuscaloosa, AL, USA.