Dual role of autophagy in HIV-1 replication and pathogenesis

Table 2 General methods for measuring autophagy*

Targeted component of autophagy	Procedure **
Direct enumeration and quantitation of autophagosomes. Visible as double-membrane vesicles.	Electron microscopy***
LC3-II to LC3-I ratio. Provides a measurement of autophagic flux with the LC3B-II/LC3B-I ratio concomitantly increasing with autophagosome numbers.	WB****
LC3 localization. Punctate spots visible by microscopy. Total intracellular levels may increase along with autophagosome numbers.	ICC, FC, transfection of LC3 reporter plasmid followed by fluorescent microscopy or FC
Quantitation of autophagy-associated gene expression levels, e.g., BECN1.	qPCR
Quantitation of autophagy-associated protein levels, e.g., Beclin-1.	WB, ELISA
Silencing of autophagy-associated genes.	RNAi
Manipulation of autophagic flux.	Use of rapamycin, bafilomycin A1, and 3-MA

* Because the process is highly conserved among eukaryotes, these methods are broadly applicable to studies of autophagy in humans and animal models[27, 31].
** Western blot (WB), flow cytometry (FC), immunocytochemistry (ICC), quantitative PCR (qPCR), RNA interference (RNAi).
*** “Gold standard” method for quantifying the number of autophagosomes.
**** “Gold standard” method for quantitating autophagic flux. Some commercially available anti-human LC3 antibodies are cross-species reactive, allowing for studies with non-human primate cells (e.g. Anti-MAP1LC3B2, cat.no. AB2970, Millipore, Billerica, MA).

ISSN: 1742-6405