Anti lc3b map1lc3b antibody

Dulbecco’s minimum essential medium Eagle (DMEM), trypsin–EDTA, 3-methyladenine (3MA) and all other reagents not specifically mentioned were of analytical grade and purchased from Sigma-Aldrich (St. Louis, USA). Streptomycin, fungizone and penicillin were manufactured by Thermo Fisher Scientific (Massachusette (MA), USA). Crystal violet, gluteraldehyde and triton X-100 were purchased from Merck (Darmstadt, Germany). Anti-LC3B/MAP1LC3B-antibody was supplied by Novus Biological [Littleton, Colorado (CO), USA].

To detect autophagy, flow cytometry was used employing microtubule-associated protein 1A/1B-light chain 3 (LC3) using a conjugated rabbit polyclonal anti-LC3B antibody which can be detected in FL1 (excitation = 488 nm; emission = 525 nm) [33 (link)]. After 24 h of exposure to ESE-15-ol with or without 3MA, washed trypsinized cells were resuspended in ice-cold PBS containing 0.01% formaldehyde and samples were incubated for 10 min at 4 °C. Cells were centrifuged and resuspended in 200 μl PBS to which 1 ml ice-cold 100% methanol was added and incubated for 15 min at 4 °C. Cells were washed twice with PBS. Cell pellet was resuspended in 200 μl PBS containing 0.05% triton X-100, 1% BSA, 40 μg/ml PI and anti-LC3B/MAP1LC3B-antibody (1:200) (Novus Biological, Littleton, CO, USA) and incubated for 2 h at 4 °C. Cells were washed twice with 1 ml PBS containing 0.05% triton X-100 and 1% BSA. LC3 fluorescence was measured in flow channel 1 (FL1) on the FC500 system flow cytometer (Beckman Coulter, California, USA). Three biological repeats with a minimum of 10,000 cells were used and data was analyzed with Cyflogic flow cytometry analysis software (Beckman Coulter, California, USA). Any cell debris and cell clumps were removed from analysis.