LC3B Antibody Kit for Autophagy

Protocol tips

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	Dilute the LC3B rabbit polyclonal antibody in blocking buffer to prepare 0.5 μg/mL working solution.

Protocol tips
Dilute the LC3B rabbit polyclonal antibody in blocking buffer to prepare 0.5 μg/mL working solution.

Publication protocol

Jurkat and K562 cells with or without treatment were pelleted and resuspended in 100 µl of Solution A fixative for 15 min at room temperature (RT; cat. no. GAS‐002A‐1, Caltag, UK). Cells were then washed in PBS buffer. Cell pellets were then permeabilized with 0.25% Triton X‐100 (cat. no. X100–500ML, Sigma Chemicals) for 15 min at RT. Cells were washed in PBS buffer. Anti‐LC3B polyclonal antibody (0.25 µg; cat. no. L10382, Invitrogen) or rabbit immunoglobulin (0.25 µg; cat No I5006, Sigma Chemicals) was used as an isotype control and incubated for 0.5 h at RT. Cells were then washed in PBS buffer. Cells were then labeled with 0.125 µg of secondary fluorescent conjugate Alexa Flour 647 goat anti‐rabbit IgG (cat. no. A21244, Invitrogen) for 30 min at RT. Cells were then washed in PBS buffer and resuspended in 400 µl of PBS in the presence of DNA viability dye, DAPI (200 ng/ml), see Supplementary Information figure 2. Analysis of LC3B‐Alexa Fluor647 signal was achieved by determining the MFI of the whole histogram signal for previously live cells gated from a FSC versus SSC dot‐plot. LC3B MFI upregulation from autophagy‐induced samples and untreated cells were compared to show the degree of induced autophagy. Average fold increase for autophagic samples was calculated from average control MFI LC3B levels and the individual data points shown (n = 3). LC3B‐labeled samples were also compared to corresponding isotype control samples in an overlaid histogram. About 10,000 events were collected by flow cytometry.

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