Mitrotracker red cm h2x ros

SH-SY5Y cells were seeded on 12 mm poly-D-lysine coated glass coverslips (BD Biosciences, Franklin Lakes, NJ) at a density of 7 × 10⁴ cells/coverslip and allowed to attach for 1 day prior to 6 day differentiation in DMEM (Mediatech) with 10% FBS and 10 μM retinoic acid (Sigma). Cells were subsequently treated with 50 μM ADan pE or ADan E for up to 24h, fixed with 4% paraformaldehyde, and blocked with 20 mg/ml BSA in PBS containing 0.3% Triton X-100. After incubation with mouse monoclonal anti-cyt c antibody (BD Biosciences; 1:200 in PBS containing 5 mg/ml BSA, 2h at RT) followed by Alexa Fluor 488-conjugated anti-mouse IgG (Life Technologies; 1:200 in PBS with 5 mg/ml BSA, 1h at RT), nuclei were counterstained with TO-PRO-3 iodide (Life Technologies; 1:1,000) as previously described [36 (link)-38 (link)]. To examine cyt c mitochondrial localization in conjunction with changes in the membrane potential of the organelle, cells –after peptide treatment– were washed with warm PBS and incubated for 30 minutes with 1.5 μM Mitrotracker Red CM-H₂X Ros (Life Technologies), followed by cyt c immunostaining as above. All images were acquired using a Zeiss LSM 510 microscope and analyzed using Image J.

SH-SY5Y cells were seeded on 12 mm poly-D-lysine coated glass coverslips (BD Biosciences, Franklin Lakes, NJ) at a density of 7 x 10⁴ cells/coverslip and allowed to attach for 1 day prior to treatment with 50 μM ABri pE/E, Bri1-23 pE/E, or Aβ42 for 4–16 hours. After washing once with cold PBS, cells were fixed with 4% paraformaldehyde and blocked with 20 mg/ml BSA in PBS containing 0.3% Triton X-100. Cells were subsequently incubated with mouse monoclonal anti-cyt c antibody (BD Biosciences; 1:200 in PBS containing 5 mg/ml BSA, 2h at RT) followed by Alexa Fluor 488-conjugated anti-mouse IgG (Life Technologies, Carlsbad, CA; 1:200 in PBS with 5 mg/ml BSA, 1h at RT), and nuclei were counterstained TO-PRO-3 iodide (Life Technologies; 1:1,000 in PBS, 10 minutes at RT) as previously described. To examine mitochondrial localization in conjunction with changes in the membrane potential of the organelle, cells–after peptide treatment–were washed once with warm PBS and incubated for 30 minutes with 1.5 μM Mitrotracker Red CM-H₂X Ros (Life Technologies), followed by cyt c immunostaining as above. All images were acquired using a Zeiss LSM 510 confocal microscope and analyzed using Image J (NIH, Bethesda, MD; http://rsbweb.nih.gov).