Anti-LC3B antibody produced in rabbit

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	For immunostaining, incubate cells with primary Ab overnight at 4°C

Protocol tips
For immunostaining, incubate cells with primary Ab overnight at 4°C

Publication protocol

Western blot of LL-37, BECN1, ATG5, LC3, and SQSTM1
Expression of LL-37 and markers of autophagy were evaluated by western blot analysis. Cellular extracts and culture supernatants were used for western blot analysis. When macrophages and THP-1 cells were infected with Mtb, the cellular extracts were prepared according to the manufacturer protocol for protein extraction by using Trizol reagent (Ambion, Life Technologies, 10296010). In some experiments, cellular extracts were prepared by using radioimmunoprecipitation assay buffer (Sigma, R0278). Cell culture supernatants were enriched for polypeptides on OASIS 1cc reversed-phase HLB columns (Waters Corporation, 186003908). The proform of LL-37 (hCAP-18) and the active form LL-37, were detected by monoclonal anti-LL-37.33 Autophagy-related protein BECN1 and ATG5 were detected by rabbit anti-BECN1 (Sigma, B6186) and rabbit anti-ATG5 (Sigma, A0731). The cytosolic inactive form (LC3-I) and lipidated form (LC3-II) of the autophagy marker LC3, were detected by rabbit anti-LC3 (Sigma, L7543). Furthermore, an additional marker of autophagy, SQSTM1 expression, was also detected by rabbit anti-SQSTM1 (Sigma, P0067) through western blot analysis. The western blot images were acquired by Odyssey LI-COR Imaging Systems (LI-COR, Lincoln, Nebraska, USA).

Fluorescence microscopy
For immunostaining, human MDMs and THP-1 cells were seeded on 8-well chamber slides (2 × 105 cells/well) and were differentiated by M-CSF and phorbol myristate acetate, respectively. The cells were infected with the virulent Mtb H37Rv for 4 h and stimulated with PBA, and/or 1,25(OH)2D3, or rapamycin or LL-37 for 24 h. After that cells were fixed with 4% paraformaldehyde in PBS at room temperature (RT) for 10 to 15 min and permeabilized with 0.1% Triton X-100 (Sigma, T9284) and 0.1% sodium citrate in PBS for 5 min. Then the cells were treated with 10% goat serum (Jackson ImmunoResearch, 005–000–121) for one h at room temperature and stained with primary antibodies, including mouse anti-human LC3 (MBL International, M152–3) or rabbit anti-LC3 (Sigma, L7543), rabbit anti-SQSTM1 (Sigma, P0067) and mouse anti-human LL-37,33 overnight at 4°C. After washing to remove excess primary antibodies, the cultures were incubated for one h at room temperature with the following fluorescently labeled secondary antibodies: anti-mouse IgG-Alexa Fluor 488 or 594 and anti-rabbit IgG-Alexa 594 or 488 (Jackson Immunoresearch, 115–545–062, 115–585–062, 111–585–045, 111–095–144), and then washed 3 times with PBS. Cells were loaded with DAPI mounting media (Vector Laboratories, H-1200) for visualization of nuclei, and imaged with an Olympus confocal microscope (Olympus FluoView™ FV1000 confocal microscope, Melville, NY, USA). Quantification of autophagy was performed based on the percentage of the cells with LC3-II punctate dots by ImageJ software. For positive control of autophagy activation, cells were incubated in complete medium that contained 100 nM rapamycin (Sigma, R0395) for 24 h.

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