The sections were washed in distilled water, dehydrated and coverslipped with neutral balsam

The sections were washed in distilled water, dehydrated and coverslipped with neutral balsam. dispersed with a 200mu mesh under sterile condition. The spleen cells were mixed with the myeloma cell line Sp2/0-Ag14 (ATCC, CRL-1581) at a 10:1 ratio, while fusion reagent Polyethylene Glycol 1500 solution (Roch 783641) was added dropwise into the mixture. After plating the cell mixture to microwell plates, the hybridomas were selected by adding HAT Supplement (Gibco, 31062C011) to the medium. Anti-A1C42 antibody secreting clones were screened and subcloned based on the reaction in full length human A1C42 coated ELISA plates. The isotype of Lomustine (CeeNU) the clone 3F5, as determined using Lomustine (CeeNU) the Mouse Typer Isotyping Kit (Bio-Rad, 17C2055), is IgG2b Kappa. The antibody was purified using Gammabind Plus Sepharose (GE Healthcare, 17-0886-02). ELISA plates coated with different human A peptides were used to analyze the reactivity of mAb clone 3F5. Measurement of antigen-antibody affinity and specificity Synthetic A1C42 (100 L, American Peptide Company, California, USA) (100 ng/mL) was added to the wells in 96-well ELISA plates (Corning, USA). The samples were Lomustine (CeeNU) coated at 4C overnight. The plates were washed and blocked using 5% Lomustine (CeeNU) bovine serum album (BSA) in 5% CO2 atmosphere at 37C for 2 h. Samples were then naturally dried for future use. Thereafter, synthetic A1C42 (1 g/mL) without conjugation was diluted (1, 0.5, 0.25, 0.125, 0.0625, 0.03125 and 0 g/mL) and then pre-incubated with 3F5 (0.05 g/mL) at 4C overnight. The mixtures were added in A1C42 pre-coated ELISA plates for 90 min at 37C to measure the competitive binding by 3F5 with free A1C42 or coated A1C42. The optical density (OD) was detected at 495 nm on a microplate reader (BIO-RAD Model 2550 EIA Reader, USA). Classical peptide mapping using binding ELISA was performed to identify the epitope of A1C42 recognized by 3F5. The 96-well ELISA plates were coated with different A1C42 fragments (aa1-42, aa1-11, aa12-28, aa25-35, aa33-42), and 3F5 was then Rabbit Polyclonal to DCLK3 incubated with A1C42 fragments for 72 h at 37C. 3, 3, 5, 5 -Tetramethylbenzidine (TEM) was added as a substrate in each well and optical density (OD) was detected at 450 nm on a microplate reader (BIO-RAD). Neurite outgrowth assay Neurite outgrowth was examined according to previously described method [8]. Briefly, 2103 SH-SY5Y cells/well were seeded in a 24-well plate and cultured for 24 h at 37C in a 5% CO2. 10 M all-trans-retinoic acid (RA) were co-incubated with SH-SY5Y cells for 5 days followed by incubation with 10 M A1C42 fibrils. 3F5 antibody (10 g/mL and 20 g/mL) and IgG (10 g/mL) were then added into the plate for an additional 24 h. The supernatant was discarded and 200 L 4% paraformaldehyde was added to each well to fix the cells. Cell images were acquired by an inverted microscope (Olympus, Japan) and 10 fields (100) were analyzed per group. MTT assay SH-SY5Y neuroblastoma cells were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Methyl thiazolyl triumvirate (MTT) assay was used to measure the ability of 3F5 to reduce the cytotoxicity of A1C42 fibrils. Briefly, SH-SY5Y cells were grown on 96-well plates at a density of 4105 for 24 h. After treatment with the different concentrations of 3F5 (40 g/mL, 20 g/mL, 10 g/mL and 5 g/mL) and 10 M A1C42 for 48 h, the cells were incubated with 10 L MTT Lomustine (CeeNU) (Sigma,.

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