LC3A/B (D3U4C) XP® Rabbit mAb

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Gives distinct bands corresponding to LC3I and LC3II	1:1000 dilution for primary antibody

Upstream tips
Gives distinct bands corresponding to LC3I and LC3II
Protocol tips
1:1000 dilution for primary antibody

Publication protocol

For F-actin staining 4 × 104 cells were seeded, treated with 2.5 μM PP242, 500 nM wortmannin or 1 μM rapamycin and cultured for 3 days (U87MG) and 7 days (GL15 and U251). Cells were extensively washed with PBS, fixed at room temperature with 4% paraformaldehyde in PBS for 20 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min and washed three times with PBS. After overnight incubation with blocking buffer (3% bovine serum albumin [BSA], 1% glycine in PBS), cells were incubated at room temperature with TRITC-phalloidin (1:250; Sigma Aldrich®) in PBS for 30 min, washed three times with PSB 0.1% Tween and two times with PBS, incubated with 4’,6-diamidino-2-phenylindole (DAPI; 2 μg/ml; Sigma Aldrich®) for 30 s washed two times with PBS and air dried. For LC3 detection 4 × 104 cells were seeded, treated with 2.5 μM PP242, 500 nM wortmannin or 1 μM rapamycin and cultured for 24 h on glass coverslips. Cells were extensively washed with PBS, fixed at room temperature with cold methanol for 7 min, permeabilized with 0.1% Triton X-100 in PBS for 5 min and washed three times with PBS. After overnight incubation with blocking buffer (3% BSA, 1% glycine in PBS), cells were subjected to immunofluorescence using a monoclonal anti LC3A/B (1:50; Cell Signaling Technologies) and a goat anti-rabbit TRITC (1:50; Sigma Aldrich®) antibodies.

For Bromodeoxyuridine (BrdU) incorporation assay 4 × 104 cells were seeded, treated with 2.5 μM PP242, 500 nM wortmannin or 1 μM rapamycin and cultured for 24 h or 72 h on glass coverslips. Ten micrometer BrdU (Sigma Aldrich®) was added to the culture medium 1 h before fixation with cold methanol at −20°C. Cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min, incubated with 2N HCl for 30 min, washed three times with sodium borate buffer and once with PBS. Then, cells were subjected to immunofluorescence using a monoclonal anti-BrdU (1:50; Santa Cruz Biotechnology®) and Alexa fluor® 488 donkey anti-mouse (1:50; Life Technologies) antibody. BrdU-positive cells and total cells, counterstained with DAPI, were counted and the BruU-positive cells/total cells ratio was calculated. For comparative analysis, pictures were taken at a constant exposure time and gain in the same experimental setting, and ten random fields/coverslip were photographed. Coverslips were mounted and the preparations were viewed in a DMRB Leica epimicroscope equipped with a digital camera.

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for LC3A/B (D3U4C) XP® Rabbit mAb below.

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