Tween 20

A solution of 10 mM phosphate-buffered saline (pH 7.4, PBS) supplemented with 0.05% (v/v) surfactant Tween-20 (Thermo Fisher Scientific, Waltham, MA, USA) served as the wash solution (PBST). A buffer for serum dilution and blocking (Buffer B) was prepared by adding SuperBlock blocking solution (10 v/v%, Thermo Fisher Scientific/Pierce, Waltham, MA, USA) and Tween 20 (0.05 v/v, % Thermo Fisher Scientific, Waltham, MA, USA) to 10 mM (pH 7.4, PBS). Bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA) solution (1 w/v%) was prepared in PBST. HRP-labeled goat anti-bovine immunoglobulin G (H+L) obtained from Jackson ImmunoResearch (West Grove, PA, USA) was diluted in Buffer B immediately before use. Potassium ferrocyanide {K₄[Fe(CN)₆]} and potassium ferricyanide {K₃[Fe(CN)₆]} purchased from Thermo Fisher Scientific/ACROS Organics (Waltham, MA, USA) were stored dry and prepared fresh before each experiment. Purified HRP enzyme and 3,3′, 5,5′-tetramethylbenzidine (TMB) substrate were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Other chemicals such as acetone and 2-propanol were purchased from Thermo Fisher Scientific/Fisher Chemical (Waltham, MA, USA).

LSK-SLAM cells were sorted as described above. Cells were cultured for 24 h as described above, then fixed in 4% paraformaldehyde in PBS on glass slides for 30 min at room temperature. Cells were washed twice in PBS, then permeabilized in 0.5% Triton X-100 (ThermoFisher) in PBS for 10 min at room temperature. Cells were then washed twice in PBS and blocked in 10% bovine serum albumin (BSA; Sigma-Aldrich) in PBS. Cells were stained with rabbit anti-LC3 (1:500; MBL) in 5% BSA in PBS supplemented with 0.1% TWEEN 20 (Sigma-Aldrich) for 1 h at room temperature and then overnight at 4°C. After primary stain, cells were washed four times in PBS with 0.1% TWEEN 20. Secondary stain was donkey anti-rabbit IgG conjugated to Alexa Fluor 555 (1:500; ThermoFisher) in 5% BSA in PBS supplemented with 0.1% TWEEN 20, incubated for 1 h at room temperature and protected from light, followed by four washes in PBS with 0.1% TWEEN 20. Cells were counterstained with DAPI (1μg ml^-1) in PBS for 5 min at room temperature and then washed twice in PBS. Slides were mounted with ProLong Gold antifade reagent (Invitrogen) and cured at room temperature for at least 24 h. Slides were imaged on an Olympus IX81 fluorescent microscope setup with a Hamamatsu Camera Controller C10600. Images were recorded and analyzed using the MetaMorph for Olympus Advanced software.