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Detection of autophagy by Flow Cytometry




The LC3 is a marker protein for the mammalian autophagy. The C-terminal fragment of LC3 is cleaved immediately after LC3 synthesis to yield a cytosolic form called LC3-I. A subpopulation of LC3-I is further converted to a form, i.e., LC3-II, that located on the autophagosomal membrane.

Nutrient starvation increases the levels of autophagy. During the progression of autophagy, autophagosomes fuse with lysosomes, and the contents along with LC3-II get degraded. Thus, the fluorescence intensity decreases in flow cytometry using this antibody.


References


Experiment conditions

Starvation: Hank's Balanced Salt Solution, 4 hr
Cell dissociation: Tripsin-EDTA
Fixation: 4% PFA (RT 15min)
Permeabilization: Digitonin (100 ug/mL) (RT 15min)
1st antibody: anti-LC3 (PM036), normal rabbit IgG, R.T. 30 min
2nd antibody: anti-rabbit IgG-FITC (IM-0833), R.T. 30 min

Atg5-/-MEF Cell line is provided by courtesy of Noboru Mizushima, M.D., Ph.D.,
Tokyo Medical and Dental University



Anti-LC3 (Human) pAb (code no. PM036) detailed information

Anti-LC3 (Human) mAb (code no. M152-3) detailed information