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All surgical and experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at Seoul National University. Immunohistochemistry was performed as previously described 30. Information on the antibodies used in this study is provided in Table 1. Luxol fast blue staining was performed to calculate lesion volume as previously described 31. Images of the stained sections were taken under microscope (Axiovert200, Carl Zeiss, Munchen, Germany) and the lesion size (x�Cy stage) was calculated using the Axiovision 4.8 image program. The total lesion volume was calculated R428 by summing individual subvolumes following the Cavalieri method 32. Apoptotic cells were detected using an Apoptaq Plus Fluorescein in situ cell apoptosis detection kit (Chemicon, Temecula, CA, USA) according to the manufacturer's instructions. Real-time RT-PCR was performed as previously described 33. ABT-737 cell line Relative mRNA levels were calculated according to the 2?����Ct method 34. All Ct values were normalized to GAPDH. All experiments were performed at least three times. The PCR primer sequences used in this study are listed in Table 2. Hind limb motor function was assessed by open field locomotion using BMS 19. All behavioral tests were performed blind. Neutrophils and monocytes were prepared as described previously 35, 36. Isolated neutrophil or monocyte cells (5��104 cells/well) were seeded onto a 5-��m pore-size Transwell plate (Costar, Corning, NY, USA) and incubated for 2?h in culture medium, in the presence or absence of CXCL1 (100?ng/mL, Bio-Research Products, South Lancaster, MA, USA) or CCL2 (50?ng/mL, Bio-Research Products) in the lower chamber. Neutrophils or monocytes that had migrated into the lower chamber were counted under magnification. In situ gelatinolytic activity was detected as described Smoothened Agonist manufacturer previously 37. Flow cytometry was performed as previously described 30. PE-conjugated anti-CD11a (eBioscience, San Diego, CA, USA), FITC-conjugated anti-CD11b (BD Pharmingen, San Diego, CA, USA), PE-conjugated anti-CD45 (BD Pharmingen), FITC-conjugated anti-Gr-1 (BioLegend, San Diego, CA, USA) or Cy5.5-conjugated anti-CXCR2 (BioLegend) antibodies were used for flow cytometry. Spinal cord tissues (��0.25?cm to the injury site) were dissected from the spinal cord and proteins were extracted using tissue lysis buffer (137?mM NaCl, 20?mM Tris-HCl, 1% NP40, and protease inhibitor cocktail set III (Calbiochem, La Jolla, CA, USA). The KC/CXCL1 in the tissue lysates was measured using ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. Data are presented as mean��SEM. Statistical analyses for real-time PCR data, flow cytometry data, cell migration assay data, ELISA data and image analysis were performed using a one-way ANOVA for measurements, followed by an independent Tukey's post-hoc test to compare the procedures. A p