Autophagy assay cell type - PC-12

As autophagy is a multi-step process which includes not just the formation of autophagosomes, but most importantly, flux through the entire system, including the degradation upon fusion with lysosomes, which makes it quite challenging for detection. There are several methods for detection in mammalian cells, including immunoblotting analysis of LC3 and p62 and detection of autophagosome formation/maturation by fluorescence microscopy, Currently, there is no single “gold standard” for determining the autophagic activity that is applicable in every experimental context, hence it is recommended to go for the combined use of multiple methods to accurately assess the autophagic activity in any given biological setting.

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Found 5 matching solutions for this experiment

Protocol tips
Lysotracker Red (Invitrogen, San Diego, CA, USA) was used to stain lysosomes and autophagolysosomes. PC-12 cells were incubated with PAMAM dendrimers G5 for a series of times, then Cyto-ID Green Dye, LysoTracker Red, and Hoechst 33342 were used to stain cells at 37 oC for 20 min. After that, cells were washed in RPMI-1640 medium and observed by confocal microscopy immediately. All the relative fluorescence intensity was measure by inverted confocal microscope software (Carl Zeiss LSM710, Carl Zeiss, Germany).
JNK1 Antibody (F-3)

Santa Cruz Biotechnology

Upstream tips
- Lyse cells in lysis buffer containing 100 mm NaCl, 50 mm Tris-HCl, 1 mm EGTA, 10 mmMgCl2, pH 7.2) containing 1 % Triton X-100, phosphatase, and protease inhibitor mixture for 5 min at room temperature and thereafter kept on ice.
Protocol tips
- Incubate the sample with the antibody for 1–12 h at 4°C, preferably under gentle agitation or rotation
LC3B (D11) XP® Rabbit mAb

Cell Signaling Technology

Protocol tips
- Incubate Primary Ab membrane overnight at 4 °C with constant shaking
Protocol tips
- incubate with primary antibodies at 4°C overnight.
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